Figure legends
Fig. 1. Phylogenetic tree of four bacterial isolates and representatives of related species based on the 16S rRNA gene sequences. The tree was inferred from an alignment of 1,380-bp-long sequences of 16S rRNA genes, and constructed by the neighbor-joining method. The numbers at the nodes denote the bootstrap percentages derived from 1,000 replications. Scale bar: 0.01 substitutions per nucleotide position.
Fig. 2. Anaerobic growth of P. denitrificansMIC1-1T (A), P. denitrificans AT004 (B),P. denitrificans KGS048 (C), Prolixibacter sp. SD074 (D).Prolixibacter sp. NT017 (E), and
P. bellariivorans JCM 13498T (F). These strains were grown anaerobically in either YSw medium (open symbol) or ammonium-free YSw medium (closed symbol) in the presence (circle) or absence (triangle) of 10 mM sodium nitrate. Data represent the means (n = 3), with standard deviation values less than 18% of the corresponding mean values.
Fig. 3. Scanning electron micrographs showing the surface and cross-sections of Fe0 foils incubated anaerobically with nitrate-reducing Prolixibacter strains. Fe0 foils were incubated for 30 days in corrosion-test medium in the presence of P. denitrificansMIC1-1T (A–C), P. denitrificans AT004 (D–F),P. denitrificans KG048 (G–I), and Prolixibacter sp. SD074 (J–L). A, D, G, and J: the surface of Fe0 foils (×300 magnification). B, E, H, and K: the surface of Fe0foils (×1,000 magnification). C, F, I, and L: the cross-section of Fe0 foils (×300 magnification). Scale bar: 10 µm for A, C, D, F, G, I, J, and L; 1 µm for B, E, H, and K. Scanning electron micrographs were obtained from two independent experiments.
Fig. 4. Scanning electron micrographs showing the surface and cross-sections of Fe0 foils incubated anaerobically with nitrate-non-reducing Prolixibacter strains. Fe0 foils were incubated for 30 days in corrosion-test medium in the presence of Prolixibacter sp. NT017 (A–C), andP. belleriivorans JCM 13498T (D–F). The aseptic controls are also shown (G–I). A, D, and G: the surface of Fe0 foils (×300 magnification). B, E, and H: the surface of Fe0 foils (×1,000 magnification). C, F, and I: the cross-section of Fe0 foils (×300 magnification). Bar = 10 µm for A, C, D, F, G, and I. Scale bar: 1 µm for B, E, and H. Scanning electron micrographs were obtained from two independent experiments.
Fig. 5. Corrosion potential and corrosion current of an Fe0 electrode immersed in the cultures of P. denitrificans strain MIC1-1T and Prolixibactersp. NT017. Corrosion potential and corrosion current of an Fe0 electrode were determined as described in the Materials and Methods. (A) Change in the corrosion potential of an Fe0 foil used as the working electrode. (B) Change in the corrosion current generated at the Fe0 foil poised at 25 mV versus corrosion potential. Solid red line: in the presence ofP. denitrificans MIC1-1T; dashed green line: in the presence of Prolixibacter sp. strain NT017; dotted black line, aseptic control.
Fig. 6. Nitrate reduction and the accumulation of nitrite and ammonium in the cultures of nitrate-reducing Prolixibacterstrains. Nitrate-reducing Prolixibacter strains were grown under an atmosphere of N2:CO2 (4:1) in corrosion-test medium either in the presence (A to E) or absence (F to J) of an Fe0 foil. In F to J, the concentrations of oxidized iron in the aseptic controls were between 0 mM at day 0 and 0.2 mM at day 28. The strains used were P. denitrificansMIC1-1T (A and F), P. denitrificans AT004 (B and G), P. denitrificans KGS048 (C and H) Prolixibactersp. SD074 (D and I), and aseptic control (E and J). Filled circle: nitrate concentration, filled triangle: nitrite concentration, cross: ammonium concentration, and open circles: concentration of oxidized iron. Data represent means (n = 3), with standard deviation values less than 8.9% of the corresponding mean values.
Fig. 7. The relationship between Fe0oxidation and nitrite reduction. The data in Fig. 6 are rearranged to show the relationships between the increment in the concentration of oxidized iron (open circles) and the consumed concentration of nitrite (filled triangle) during three weeks after day 7. The latter concentration was calculated as described in the text. The strains used were P. denitrificans MIC1-1T (A), P. denitrificans AT004 (B), P. denitrificans KGS048 (C), andProlixibacter sp. SD074 (D). Bold lines were obtained by multiplying the values of the consumed concentration of nitrite bya where a = 1.25 for A, 2 for B, 2.4 for C, and 1.85 for D. Data represent means (n = 3), with standard deviation values less than 8.9% of the corresponding mean values.
Table 1. Bacterial strains used in this study.