Iron-corroding methanogen isolated from a crude-oil storage tank

Abstract

Microbiologically influenced corrosion of steel in anaerobic environments has been attributed to hydrogenotrophic microorganisms. A sludge sample collected from the bottom plate of a crude-oil storage tank was used to inoculate a medium containing iron (Fe(0)) granules, which was then incubated anaerobically at 37 degrees C under an N(2)-CO(2) atmosphere to enrich for microorganisms capable of using iron as the sole source of electrons. A methanogen, designated strain KA1, was isolated from the enrichment culture. An analysis of its 16S rRNA gene sequence revealed that strain KA1 is a Methanococcus maripaludis strain. Strain KA1 produced methane and oxidized iron much faster than did the type strain of M. maripaludis, strain JJ(T), which produced methane at a rate expected from the abiotic H(2) production rate from iron. Scanning electron micrographs of iron coupons that had been immersed in either a KA1 culture, a JJ(T) culture, or an aseptic medium showed that only coupons from the KA1 culture had corroded substantially, and these were covered with crystalline deposits that consisted mainly of FeCO(3).

FIG. 1.

Photomicrographs of microorganisms present after five successive transfers in an enrichment culture for which iron was the sole source of electrons. (A) Phase-contrast micrograph of cells in the enrichment culture. (B) Fluorescence micrograph of the sample shown in panel A. (C) Phase-contrast micrograph of a small corrosion particle. (D) Fluorescence micrograph of the particle shown in panel C. The microbes that autofluorescence at 420 nm (F₄₂₀) are methanogens. Bar = 5 μm.

FIG. 2.

Cell count, iron dissolution, hydrogen production, and methane production. All experiments were carried out with iron granules as the sole source of electrons. (A) Cell count; (B) ferrous iron concentration in the supernatants of centrifuged cultures; (C) hydrogen production; and (D) methane production. Open squares, enrichment culture; filled squares, enrichment culture supplemented with 20 mM BESA; filled circles, strain KA1 culture; open circles, strain JJ^T culture; filled diamonds, aseptic control. Data points and bars are the means and standard deviations, respectively (n = 4).

FIG. 3.

Scanning electron micrographs of the surface and cross-section of iron coupons present in strain JJ^T and strain KA1 cultures. (A) The surface of an iron coupon incubated for 2 weeks in a JJ^T culture. (B) The cross-section of an iron coupon incubated as for panel A. (C) The surface of an iron coupon incubated for 2 weeks in a KA1 culture. (D) The cross-section of an iron coupon incubated as for panel C.