Accurate mapping of the composition and ultrastructure of minerals and cells is key to understanding biogeochemical process in contaminated environments. To this end we developed two LN2-based apparatus that allow correlation of cryogenic transmission electron microscopy (TEM), synchrotron hard X-ray microprobe (SHXM) and scanning transmission X-ray microscopy (STXM) datasets. These cryogenic methods allowed the precise determination of the distribution, valence state and structure of selenium in intact biofilms directly flash-frozen in the field, during a biostimulation experiment in a contaminated aquifer near Rifle, CO, USA. Results were replicated in the laboratory via anaerobic selenate-reducing enrichment cultures. 16S rRNA analyses of field-derived biofilm indicated the dominance of Betaproteobacteria from the Comamonadaceae family, and uncultivated members of the Simplicispira genus. Correlative analyses of the cultures provided direct evidence for microbial dissimilatory reduction of Se(VI) to Se(IV) to 25-300 nm diameter red amorphous Se0 aggregates. Cryogenic X-ray diffraction and Se K-edge extended X-ray absorption fine structure spectroscopy revealed red amorphous Se0 with a first shell Se-Se interatomic distance of 2.339 ± 0.003 Å. Cryo-STXM showed that these aggregates are strongly associated with a protein-rich biofilm matrix containing acidic polysaccharides. From Rifle groundwater, we isolated a selenate reducing anaerobe that shares 98.9% 16S rRNA gene sequence identity with Dechloromonas aromatica RCB. We refer to this isolate as Dechloromonas selenatis strain RGW99. 3D cryo-electron tomography showed that Se0 particles do not form inside the cytoplasm but rather originate in the cell membrane. The end product of selenate reduction by D. selenatis is 240 ± 66 nm diameter red amorphous Se0 colloidal aggregates, found to be stable for months. Overall, these results established a role for D. selenatis RGW99 in selenate reduction in the Rifle aquifer and provided new insights into the nature and stability of selenium bioreduction products in the subsurface.

Correlative cryo-TEM, cryo-SHXM and cryo-STXM study of selenium bioreduction in a contaminated aquifer

251st National Meeting (2016)

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