Electron Microscopy Evaluation of the Role of Dissimilatory Metal-reducing Bacteria in Biomineralization Pathways

The importance of microorganisms in the biogeochemical cycling of Fe is well-recognized [1]. Dissimilatory metal-reducing bacteria (DMRB), which are ubiquitous in soils and aquifers, couple the oxidation of organic matter or H2 with the reduction of various Fe(III) oxide phases to obtain energy for growth and function. They can also catalyze Fe(III) reduction under anaerobic conditions, utilizing crystalline and poorly crystalline iron oxides as a terminal electron acceptor. Microbially induced Fe mineral transformations were examined using the Shewanella putrefaciens, strain CN32 in an artificial groundwater medium in columns under advective flow conditions. Columns were filled with ferrihydrite-coated quartz sand inoculated with S. putrefaciens (initial cell density 10? mL-?). Lactate was added as an electron donor. Changes in microbial metabolism, aqueous chemistry, and solid phase distributions were monitored at time points until termination of the column experiment at 16 days [2]. Transmission (TEM) and scanning electron microscopy (SEM) was used for investigating mineral association with bacterial cells, crystal size, morphology, and spatial relationships. A special TEM sample preparation protocol developed in our laboratory was used for the accurate preservation of both the biological and mineral portion of the sample [3]. To eliminate the anaerobic sample exposure to oxygen, the whole embeddingmore » procedure, as well as the thin sectioning on an ultramicrotome was carried out in an anaerobic glove box (95% argon, 5% hydrogen). Ultra thin sections of the material were studied using a JEOL 2010 TEM operating at 200 kV coupled to an Oxford EDS system. Images were collected and analyzed using a Digital Micrograph (Gatan). Selected area diffraction patterns were evaluated by the Desktop Microscopist (Lacuna) software. Visual changes in the solid-phase within the column were evident: initial orange ferrihydrite started turning brown, and further darkened over the course of the experiment, as it was converted to predominantly goethite and magnetite [Fig. 1A, 1B]. In addition to spectroscopic methods, the presence of goethite and magnetite was further confirmed by TEM and SEM, and the spatial orientations and particle size of mineral particles were determined. Typical needle-like structures of goethite crystals were predominantly associated with the surface of ferrihydrite, but were also found coupled with microbial cell surfaces. In fact, some bacterial cells appeared completely encrusted in goethite, most likely, a result of electrostatic attraction between newly precipitated goethite and the microbial surface. Magnetite, on the other hand, was mainly associated with the ferrihydrite surface, and only rarely with the cell surface. Thus, the bacterial cell is only indirectly (by ferrous iron production) responsible for goethite and magnetite formation. Although intracellular precipitation of iron oxides in S. putrefaciens was recently reported [4], only extracellular precipitation was observed in this experimental setup. The ability of bacteria to shed the mineral deposits from their outer membranes in order to prevent their surfaces passivation caused by mineral sorption will be discussed. Bacteria appeared to primarily serve as an Fe(II) source for the system; secondary mineralization was confirmed as a function of initial Fe(II) concentrations. The mechanism of the enzymatic reduction is not completely understood, and the accountable protein functions are being intensively investigated by several molecular biology techniques. Current and future studies will include immunogold labeling at the electron microscopy level as a method for determining the localization of these proteins.« less