Arsenic (As) is a poisonous metalloid that is persistent in many soils and aquifers worldwide. This element has a high affinity for iron (Fe) and sulfur (S) minerals, and in anoxic environments As is thought to be mobilised through the oxidation and reduction of these minerals, where bacterial metabolisms are directly involved. In these conditions some bacteria derive energy from arsenate [As(V)] respiration, releasing arsenite [As(III)], the more toxic and mobile As oxyanion; other bacteria catalyse the reverse reaction, where As(III) is used as an electron donor and oxidised to the least mobile As(V). Electron microscopy and metagenomics-proteomics are the tools conventionally used to study these systems, however, they are limited to spatially correlate metabolically active microorganisms. Thus, the bacterial mechanisms of As and Fe oxidation/reduction remain poorly understood. In this project, model As and Fe reducing/oxidising bacteria were analysed using nanoscale secondary ion mass spectrometry (NanoSIMS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) for the first time. These techniques were selected due to their high spatial resolution, high sensitivity and accurate isotope ratio analysis, where stable isotope probing was used to image metabolically active bacteria using NanoSIMS. The model As(V) and Fe(III)-reducing bacteria, Geobacter sulfurreducens and Shewanella sp. strain ANA-3, were assessed in order to evaluate As mobilisation from sorbed As(V) on an Fe(III)-oxyhydroxide mineral. Scanning electron microscopy (SEM) and NanoSIMS allowed the inference of the predominant extracellular electron transport (EET) mechanisms, where G. sulfurreducens requires direct cell-mineral contact, in contrast to S. ANA-3 that uses flavins as electron shuttles. Additionally, a multistep As mobilisation mechanism was proposed for S. ANA-3. ToF-SIMS was further used for the identification of key molecules involved in the EET process in S. ANA-3, although single cells could not be imaged and biomarkers, for instance from flavins, were scarcely detected. Furthermore, two recently isolated denitrifying As(III) and Fe(II)-oxidising bacteria, Acidovorax sp. strain ST3 and Paracoccus sp. strain QY30, were studied as well as their biomineral products. In these systems high concentrations of As(III) were removed via the formation of Fe(III) precipitates, and quantitatively analysed using transmission electron microscopy and energy dispersive X-ray spectroscopy (S/TEM-EDS) complementary to NanoSIMS, SEM and X-ray diffraction analysis (XRD). Periplasmic encrustation and cell surface coating with Fe minerals were also identified. Moreover, single cells of both As-Fe reducing/oxidising systems were depth-profiled in NanoSIMS, where the subcellular As and Fe spatial distributions were imaged and modelled in 3D. This multi-technique approach successfully analysed active As and Fe respiring bacteria at the nanoscale, and NanoSIMS could rapidly become an established imaging tool used in Geomicrobiology and diverse redox systems.
|Date of Award||1 Aug 2020|
- The University of Manchester
|Supervisor||Ian Lyon (Supervisor), Jonathan Lloyd (Supervisor) & Katie Moore (Supervisor)|
- Nitrate dependent Fe(II) oxidation
- As(V) respiration
- Fe(III) respiration