Microbial reduction of Fe(III) in acidic sediments: isolation of Acidiphilium cryptum JF-5 capable of coupling the reduction of Fe(III) to the oxidation of glucose

Abstract

To evaluate the microbial populations involved in the reduction of Fe(III) in an acidic, iron-rich sediment, the anaerobic flow of supplemental carbon and reductant was evaluated in sediment microcosms at the in situ temperature of 12 degrees C. Supplemental glucose and cellobiose stimulated the formation of Fe(II); 42 and 21% of the reducing equivalents that were theoretically obtained from glucose and cellobiose, respectively, were recovered in Fe(II). Likewise, supplemental H(2) was consumed by acidic sediments and yielded additional amounts of Fe(II) in a ratio of approximately 1:2. In contrast, supplemental lactate did not stimulate the formation of Fe(II). Supplemental acetate was not consumed and inhibited the formation of Fe(II). Most-probable-number estimates demonstrated that glucose-utilizing acidophilic Fe(III)-reducing bacteria approximated to 1% of the total direct counts of 4', 6-diamidino-2-phenylindole-stained bacteria. From the highest growth-positive dilution of the most-probable-number series at pH 2. 3 supplemented with glucose, an isolate, JF-5, that could dissimilate Fe(III) was obtained. JF-5 was an acidophilic, gram-negative, facultative anaerobe that completely oxidized the following substrates via the dissimilation of Fe(III): glucose, fructose, xylose, ethanol, glycerol, malate, glutamate, fumarate, citrate, succinate, and H(2). Growth and the reduction of Fe(III) did not occur in the presence of acetate. Cells of JF-5 grown under Fe(III)-reducing conditions formed blebs, i.e., protrusions that were still in contact with the cytoplasmic membrane. Analysis of the 16S rRNA gene sequence of JF-5 demonstrated that it was closely related to an Australian isolate of Acidiphilium cryptum (99.6% sequence similarity), an organism not previously shown to couple the complete oxidation of sugars to the reduction of Fe(III). These collective results indicate that the in situ reduction of Fe(III) in acidic sediments can be mediated by heterotrophic Acidiphilium species that are capable of coupling the reduction of Fe(III) to the complete oxidation of a large variety of substrates including glucose and H(2).

FIG. 1

Effect of supplemental electron donors on the formation of Fe(II). Sediment was supplemented with glucose (A), H₂ (B), or acetate (C) and incubated under an argon gas phase at 12°C. Presented are the averages (± standard deviations) of triplicate microcosms. Symbols: ●, glucose; ■, H₂; ▾, acetate; ▴, Fe(II); ▵, Fe(II) control.

FIG. 2

Electron micrographs of strain JF-5 grown in aerobic TSB medium lacking Fe(III) (A) or in Fe-TSB medium (B to F). (A, B, and D to F) Thin-section micrographs. (C) Whole cell, negatively stained with 2% phosphotungstate. Single blebs (arrowheads) and a short “chain” consisting of four blebs (small arrow) are visible on the cell periphery. (D) Cell envelope of JF-5 with features of a gram-negative cell. (E) Micrograph showing that blebs and extrusions are membrane surrounded (arrow). (F) The long extrusion which originates from the cytoplasmic membrane reveals constrictions. Arrowheads indicate single free blebs of different sizes. Bar lengths are shown in micrometers. Abbreviations: CM, cytoplasmic membrane; CW, cell wall; I, inclusion body; S, septum (division point); M, murein layer; OM, outer membrane; V, vesicle.

FIG. 3

Formation of Fe(II) and consumption of glucose by strain JF-5 incubated in Fe-TSB medium (A) and formation of Fe(II) and consumption of O₂ by strain JF-5 incubated in Fe-TSB medium supplemented with O₂ (B). O₂ and glucose were added at the indicated time intervals (arrows). Presented are the averages (± standard deviations) of triplicate experiments. Symbols: ●, glucose; ○, glucose-supplemented TSB medium lacking Fe(III); ▴, Fe(II); □, O₂.