Orenia metallireducens sp. nov. Strain Z6, a Novel Metal-Reducing Member of the Phylum Firmicutes from the Deep Subsurface

Abstract

A novel halophilic and metal-reducing bacterium, Orenia metallireducens strain Z6, was isolated from briny groundwater extracted from a 2.02 km-deep borehole in the Illinois Basin, IL. This organism shared 96% 16S rRNA gene similarity with Orenia marismortui but demonstrated physiological properties previously unknown for this genus. In addition to exhibiting a fermentative metabolism typical of the genus Orenia, strain Z6 reduces various metal oxides [Fe(III), Mn(IV), Co(III), and Cr(VI)], using H₂ as the electron donor. Strain Z6 actively reduced ferrihydrite over broad ranges of pH (6 to 9.6), salinity (0.4 to 3.5 M NaCl), and temperature (20 to 60°C). At pH 6.5, strain Z6 also reduced more crystalline iron oxides, such as lepidocrocite (γ-FeOOH), goethite (α-FeOOH), and hematite (α-Fe₂O₃). Analysis of X-ray absorption fine structure (XAFS) following Fe(III) reduction by strain Z6 revealed spectra from ferrous secondary mineral phases consistent with the precipitation of vivianite [Fe₃(PO₄)₂] and siderite (FeCO₃). The draft genome assembled for strain Z6 is 3.47 Mb in size and contains 3,269 protein-coding genes. Unlike the well-understood iron-reducing Shewanella and Geobacter species, this organism lacks the c-type cytochromes for typical Fe(III) reduction. Strain Z6 represents the first bacterial species in the genus Orenia (order Halanaerobiales) reported to reduce ferric iron minerals and other metal oxides. This microbe expands both the phylogenetic and physiological scopes of iron-reducing microorganisms known to inhabit the deep subsurface and suggests new mechanisms for microbial iron reduction. These distinctions from other Orenia spp. support the designation of strain Z6 as a new species, Orenia metallireducens sp. nov.

Importance: A novel iron-reducing species, Orenia metallireducens sp. nov., strain Z6, was isolated from groundwater collected from a geological formation located 2.02 km below land surface in the Illinois Basin, USA. Phylogenetic, physiologic, and genomic analyses of strain Z6 found it to have unique properties for iron reducers, including (i) active microbial iron-reducing capacity under broad ranges of temperatures (20 to 60°C), pHs (6 to 9.6), and salinities (0.4 to 3.5 M NaCl), (ii) lack of c-type cytochromes typically affiliated with iron reduction in Geobacter and Shewanella species, and (iii) being the only member of the Halanaerobiales capable of reducing crystalline goethite and hematite. This study expands the scope of phylogenetic affiliations, metabolic capacities, and catalytic mechanisms for iron-reducing microbes.

FIG 1

SEM (a) and TEM (b) photomicrographs of strain Z6. The isolates used for cell morphology identification were grown in the presence of ferric citrate at 42°C in modified groundwater medium. The culture was prepared in modified groundwater medium (pH 7.0 to 7.2). Ferric citrate (10 mM) was used as the electron acceptor. H₂ (202 μmol/tube) was used as the electron donor, and acetate (5 mM) was used as the carbon source. The organism contains peritrichous pili as indicated by the enlargement of the framed area shown in the inset in panel b.

FIG 2

16S rRNA gene-based phylogenetic tree of strain Z6, phylogenetically related organisms, and previously studied representative iron-reducing organisms. Strain Z6, isolated in this study, is shown in red bold type. Previously published iron-reducing bacteria type strains are underlined, and the ones isolated from deep terrestrial subsurface are shown in red type. The NCBI accession numbers of the type strains are listed in parentheses. Sulfolobus acidocaldarius DSM 639 was used as the outgroup and is not shown. Statistical confidence for the evolutionary tree was assessed by bootstrap analysis (500 replicates) and is shown as bootstrap values in percentages. Values lower than 60 are not shown. The scale bar indicates 0.2 change per nucleotide position.

FIG 3

Effects of different geochemical factors on initial iron reduction rates and fractional amounts of ferrous iron produced. All the samples were prepared in modified groundwater medium. Ferrihydrite (10 mmol/liter) was used as the electron acceptor. H₂ (202 μmol/tube) was used as the electron donor, and acetate (5 mM) was used as the carbon source. (b and c) The culture pH was 7.0 to 7.2. Duplicate samples were prepared, and the error bars indicate standard deviation of replicate samples. The initial Fe(II) concentrations (Conc.), due to the introduction of Fe(II) from parental cultures and/or reduction by the chemical reducers (Na₂S and cysteine) present as medium components, are indicated by the dashed lines.

FIG 4

Use of different organic and inorganic substrates to support reduction of ferrihydrite by Z6. All the samples were prepared in modified groundwater medium (pH 7.0 to 7.2). Ferrihydrite (10 mmol/liter) was used as the electron acceptor. The uninoculated control samples are labeled as “Blank,” and those inoculated with strain Z6 but without electron donor are labeled as “Control.” H₂ was added as the electron donor for blank samples. Under all the other conditions, the corresponding blanks were prepared in parallel with the active cultures, and no significant changes in Fe(II) were observed. Error bars indicate average standard deviations of duplicate samples. The dashed lines show the final Fe(II) in the control to illustrate the iron reduction due to nutrient carryover from the parental cultures.

FIG 5

Reduction of different ferric iron minerals by strain Z6 (a) and relation between total (tot) mineral surface area of these minerals and fractional Fe(II) production (b). The samples were prepared in modified groundwater medium (pH 6.5). H₂ (202 μmol/tube) was used as the electron donor, and acetate (5 mM) was used as the carbon source. The total surface area was calculated by the specific surface area multiplied by the mass of the ferric iron oxide mineral used to amend each sample. All the samples were prepared in duplicate, and the error bars indicate the standard deviations of the replicates. AFH, amorphous ferrihydrite.

FIG 6

Fe K-edge EXAFS spectra and linear combination fits. Spectra from the reacted solids (red) are compared to the control reactor (blue) and the corresponding parent iron oxides at the top of each graph. The linear combination fit (black dashed line) of the control sample (blue) is shown below the comparison plot, together with the scaled components in the fit. The linear combination fit (black dashed line) of the bioreduced solids (red) is shown below the LC of the control, together with the scaled components in the fit. The numerical results of the fits are listed in Table 1.

FIG 7

Genomic reconstruction for strain Z6 based on the predicted ORFs from its genome. The solid lines indicate the metabolic functions with identified ORFs, while the dashed lines mean those without ORFs annotated in the genome of strain Z6. APS, adenosine 5′-phosphosulfate; d-fructose-6P, d-fructose 6-phosphate; acetyl-CoA, acetyl-coenzyme A; GADP, d-glyceraldehyde-3-phosphate; MECHO, acetaldehyde; EOH, ethanol; PPP, pentose phosphate pathway.