Fe-phyllosilicate redox cycling organisms from a redox transition zone in Hanford 300 Area sediments

Abstract

Microorganisms capable of reducing or oxidizing structural iron (Fe) in Fe-bearing phyllosilicate minerals were enriched and isolated from a subsurface redox transition zone at the Hanford 300 Area site in eastern Washington, USA. Both conventional and in situ "i-chip" enrichment strategies were employed. One Fe(III)-reducing Geobacter (G. bremensis strain R1, Deltaproteobacteria) and six Fe(II) phyllosilicate-oxidizing isolates from the Alphaproteobacteria (Bradyrhizobium japonicum strains 22, is5, and in8p8), Betaproteobacteria (Cupriavidus necator strain A5-1, Dechloromonas agitata strain is5), and Actinobacteria (Nocardioides sp. strain in31) were recovered. The G. bremensis isolate grew by oxidizing acetate with the oxidized form of NAu-2 smectite as the electron acceptor. The Fe(II)-oxidizers grew by oxidation of chemically reduced smectite as the energy source with nitrate as the electron acceptor. The Bradyrhizobium isolates could also carry out aerobic oxidation of biotite. This is the first report of the recovery of a Fe(II)-oxidizing Nocardioides, and to date only one other Fe(II)-oxidizing Bradyrhizobium is known. The 16S rRNA gene sequences of the isolates were similar to ones found in clone libraries from Hanford 300 sediments and groundwater, suggesting that such organisms may be present and active in situ. Whole genome sequencing of the isolates is underway, the results of which will enable comparative genomic analysis of mechanisms of extracellular phyllosilicate Fe redox metabolism, and facilitate development of techniques to detect the presence and expression of genes associated with microbial phyllosilicate Fe redox cycling in sediments.

Keywords: enrichment; iron; isolation; microbial; phyllosilicate; redox; sediment; subsurface.

Figure 1

Growth of Geobacter bremensis strain R1 with NAu-2 smectite as the electron acceptor and acetate as the electron donor. Data represent mean ± SD of triplicate cultures.

Figure 2

Fe(II) oxidation by Bradyrizobium sp. strain 22: (A) Repeated growth in aerobic FeCl2 medium; (B) aerobic oxidation of biotite (inoculum grown previously several times in aerobic biotite medium); (C) repeated growth in reduced NAu-2/nitrate medium. Data in panel A show the mean ± SD of five replicate cultures; data in panel (B) represent the results from a single culture that had been transferred several times in identical medium before conducting this experiment; data in panel (C) show the mean ± SD of triplicate cultures.

Figure 3

Growth of mixotrophic Fe(II)-oxidizing isolates on reduced NAu-2 smectite with nitrate as the electron acceptor. (A) Bradyrhizobium sp. strain in8p8; (B) Bradyrhizobium sp. strain bis5; (C) Cupriavidu necator strain A5; (D) Dechloromonas agitata strain dis5; (E) Nocardioides sp. strain in31; (F) sterile control. Data represent the mean ± SD of triplicate cultures. Symbols: •, Fe(II); ▴, nitrate; ▵, nitrite; ▿, cells.

Figure 4

Neighbor-joining tree of16S rRNA gene sequences for the isolates (circles) with the presence of the nearest reference strains and the Hanford sediment (solid triangles) and groundwater (open triangles) clones. Bootstrap values less than 50% were not shown. Numbers in parenthesis indicate the depth of the Hanford sediment sample in meter and the percentage of this clone/sequence in each clone library at the specific depth or the range of their relative abundance in groundwater pyrosequencing library.

Figure A1

Diagram illustrating the 300 Area subsurface stratigraphy, sampling strategies, and sources of microbial inocula for this study.

Figure A2

Strategy for enrichment and isolation of Fe(II)-oxidizing bacteria.

Figure A3

Photos of (A) second transfer of NAu-2 smectite-reducing enrichment from reduced Ringold sediment (8.3 mmol Fe(II) L⁻¹ in live vs. 0.3 mmol Fe(II) L⁻¹ in control; (B) primary biotite-oxidizing enrichment from oxidized Ringold material (2.3 mmol Fe(II) L⁻¹ in live vs. 10.5 mmol Fe(II) L^–1 in control; (C) second transfer of reduced NAu-2-oxidizing enrichment from oxidized Ringold material (6.0 mmol Fe(II) L⁻¹ in live vs. 12.7 mmol Fe(II) L⁻¹ in control).

Figure A4

Calvin-Benson subsystem in B. japonicum strain 22, generated by SEED Viewer version 2.0 within the RAST annotation software. Enzymes indicated in green boxes are present in the genome. Abbreviations: PRK, Phosphoribulokinase; RbcL, Ribulose bisphosphate carboxylase large chain; RbcS, Ribulose bisphosphate carboxylase small chain; PGK, Phosphoglycerate kinase; GAPDH, NAD-dependent glyceraldehyde-3-phosphate dehydrogenase; TPI, Triosephosphate isomerase; FBA, Fructose-bisphosphate aldolase; FBP, Fructose-1,6 bisphosphatase; TK, Transketolase; RPE, Ribulose-phosphate 3-epimerase; RIS, Ribose 5-phosphate isomerase.