High Resolution Imaging to Characterize the Structure and Biogeochemical Function of Microbial Biofilms Matthew J. Marshall, Alice C. Dohnalkova, David W. Kennedy, Bruce W. Arey, and James K. Frederickson Pacific Northwest National Laboratory, Richland, WA 99354 Direct examination of natural and engineered environments has revealed that the majority of microorganisms in these systems live in structured communities termed biofilms. In addition to microbial cells, biofilms are comprised of a poorly characterized organic matrix commonly referred to as extracellular polymeric substance (EPS) that may play roles in facilitating microbial interactions and biogeochemical reactions including extracellular electron transfer. In our previous communications, we have examined the composition of EPS produced by Shewanella oneidensis MR-1 during uranium [U(VI)] biotransformation and the formation of reduced, extracellular uraninite [U(IV)O 2 ] nanoparticles [1]. Using a combination of transmission electron microscopy (TEM), synchrotron-based, micro X-ray fluorescence (μ-XRF) imaging, and high-resolution immuno-TEM, a highly-hydrated 3d bacterial EPS containing redox-active, extracellular electron transfer proteins was found to be produced during microbial metal reduction [1,2]. The juxtaposition of extracellular electron transfer proteins and nanoparticulate uraninite suggested that EPS played a key role in metal capture and precipitation and, possibly, extracellular electron transfer. Therefore, understanding how biofilm EPS functions and interacts with inorganic substrates such as metal ions and mineral surfaces connects the molecular-scale biogeochemical processes to those at the microorganism-level and provides insight to how microorganisms influence larger, pore-scale biogeochemical reactions. Our current view of mineral-biofilm interactions has arisen from 2-dimensional images of 3-dimensional structures. Although a 3-dimensional structure of hydrated biofilm can readily be visualized using confocal laser scanning microscopy (CLSM), the relatively low resolution fails to resolve the fine scale interactions of EPS with minerals or newly formed nanoparticles (Fig.1). We have begun to employ cryogenic (cryo) sample processing to visualize specimens in the EM while fully preserved in their frozen-hydrated state. Preliminary experiments demonstrated that the cryoTEM produced dramatically improved cell architecture relative to the traditional TEM (Fig. 1). We have also begun to use cryoTEM to examine fully hydrated EPS. In these studies, we observed that EPS has an amorphous texture with low contrast due to sparse electron density. Notably, EPS visualization was enhanced in preliminary experiments where Shewanella cells were exposed to U(VI) (Fig. 1D). We speculate that addition of electron dense uranyl ions for bioreduction experiments provides a fortuitous contrasting agent for EPS; presumably due the binding of uranyl and/or U(IV) with EPS. We have developed a protocol for cryo-preservation and cryoSEM visualization of biofilms [2]. Our preliminary experiments demonstrated that we can successfully cryo- preserve relatively large pieces of biomass in a nearest-to-native, frozen-hydrated state. Our data suggested that the hydrated biofilm-associated EPS was significantly more extended than presumed (Fig. 2). We explored the underlying biofilm using the cryo- 260 doi:10.1017/S1431927611002170 Microsc. Microanal. 17 (Suppl 2), 2011 © Microscopy Society of America 2011 https://doi.org/10.1017/S1431927611002170 Downloaded from https://www.cambridge.org/core. IP address: 3.235.21.12, on 24 May 2020 at 09:46:06, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms.