Elucidating heterogeneous iron biomineralization patterns in a denitrifying As(iii)-oxidizing bacterium: implications for arsenic immobilization.

Anaerobic nitrate-dependent iron(ii) oxidation is a process common to many bacterial species, which promotes the formation of Fe(iii) minerals that can influence the fate of soil and groundwater pollutants, such as arsenic. Herein, we investigated simultaneous nitrate-dependent Fe(ii) and As(iii) oxidation by Acidovorax sp. strain ST3 with the aim of studying the Fe biominerals formed, their As immobilization capabilities and the metabolic effect on cells. X-ray powder diffraction (XRD) and scanning transmission electron microscopy (STEM) nanodiffraction were applied for biomineral characterization in bulk and at the nanoscale, respectively. NanoSIMS (nanoscale secondary ion mass spectrometry) was used to map the intra and extracellular As and Fe distribution at the single-cell level and to trace metabolically active cells, by incorporation of a 13C-labeled substrate (acetate). Metabolic heterogeneity among bacterial cells was detected, with periplasmic Fe mineral encrustation deleterious to cell metabolism. Interestingly, Fe and As were not co-localized in all cells, indicating delocalized sites of As(iii) and Fe(ii) oxidation. The Fe(iii) minerals lepidocrocite and goethite were identified in XRD, although only lepidocrocite was identified via STEM nanodiffraction. Extracellular amorphous nanoparticles were formed earlier and retained more As(iii/v) than crystalline "flakes" of lepidocrocite, indicating that longer incubation periods promote the formation of more crystalline minerals with lower As retention capabilities. Thus, the addition of nitrate promotes Fe(ii) oxidation and formation of Fe(iii) biominerals by ST3 cells which retain As(iii/v), and although this process was metabolically detrimental to some cells, it warrants further examination as a viable mechanism for As removal in anoxic environments by biostimulation with nitrate.

Dissimilatory Fe(III) Reduction Controls on Arsenic Mobilization: A Combined Biogeochemical and NanoSIMS Imaging Approach.

Microbial metabolism plays a key role in controlling the fate of toxic groundwater contaminants, such as arsenic. Dissimilatory metal reduction catalyzed by subsurface bacteria can facilitate the mobilization of arsenic via the reductive dissolution of As(V)-bearing Fe(III) mineral assemblages. The mobility of liberated As(V) can then be amplified via reduction to the more soluble As(III) by As(V)-respiring bacteria. This investigation focused on the reductive dissolution of As(V) sorbed onto Fe(III)-(oxyhydr)oxide by model Fe(III)- and As(V)-reducing bacteria, to elucidate the mechanisms underpinning these processes at the single-cell scale. Axenic cultures of Shewanella sp. ANA-3 wild-type (WT) cells [able to respire both Fe(III) and As(V)] were grown using 13C-labeled lactate on an arsenical Fe(III)-(oxyhydr)oxide thin film, and after colonization, the distribution of Fe and As in the solid phase was assessed using nanoscale secondary ion mass spectrometry (NanoSIMS), complemented with aqueous geochemistry analyses. Parallel experiments were conducted using an arrA mutant, able to respire Fe(III) but not As(V). NanoSIMS imaging showed that most metabolically active cells were not in direct contact with the Fe(III) mineral. Flavins were released by both strains, suggesting that these cell-secreted electron shuttles mediated extracellular Fe(III)-(oxyhydr)oxide reduction, but did not facilitate extracellular As(V) reduction, demonstrated by the presence of flavins yet lack of As(III) in the supernatants of the arrA deletion mutant strain. 3D reconstructions of NanoSIMS depth-profiled single cells revealed that As and Fe were associated with the cell surface in the WT cells, whereas for the arrA mutant, only Fe was associated with the biomass. These data were consistent with Shewanella sp. ANA-3 respiring As(V) in a multistep process; first, the reductive dissolution of the Fe(III) mineral released As(V), and once in solution, As(V) was respired by the cells to As(III). As well as highlighting Fe(III) reduction as the primary release mechanism for arsenic, our data also identified unexpected cellular As(III) retention mechanisms that require further investigation.

NanoSIMS imaging of extracellular electron transport processes during microbial iron(III) reduction.

Microbial iron(III) reduction can have a profound effect on the fate of contaminants in natural and engineered environments. Different mechanisms of extracellular electron transport are used by Geobacter and Shewanella spp. to reduce insoluble Fe(III) minerals. Here we prepared a thin film of iron(III)-(oxyhydr)oxide doped with arsenic, and allowed the mineral coating to be colonised by Geobacter sulfurreducens or Shewanella ANA3 labelled with 13C from organic electron donors. This preserved the spatial relationship between metabolically active Fe(III)-reducing bacteria and the iron(III)-(oxyhydr)oxide that they were respiring. NanoSIMS imaging showed cells of G. sulfurreducens were co-located with the iron(III)-(oxyhydr)oxide surface and were significantly more 13C-enriched compared to cells located away from the mineral, consistent with Geobacter species requiring direct contact with an extracellular electron acceptor to support growth. There was no such intimate relationship between 13C-enriched S. ANA3 and the iron(III)-(oxyhydr)oxide surface, consistent with Shewanella species being able to reduce Fe(III) indirectly using a secreted endogenous mediator. Some differences were observed in the amount of As relative to Fe in the local environment of G. sulfurreducens compared to the bulk mineral, highlighting the usefulness of this type of analysis for probing interactions between microbial cells and Fe-trace metal distributions in biogeochemical experiments.