Abstract
Certain types of anaerobic granular sludge, which consists of microbial aggregates, can reduce selenium oxyanions. To envisage strategies for removing those oxyanions from wastewater and recovering the produced elemental selenium (Se-0), insights into the microbial community structure and synthesis of Se-0 within these microbial aggregates are required. High-throughput sequencing showed that Veillonellaceae (c.a. 20%) and Pseudomonadaceae (c.a.10%) were the most abundant microbial phylotypes in selenite reducing microbial aggregates. The majority of the Pseudomonadaceae sequences were affiliated to the genus Pseudomonas. A distinct outer layer (similar to 200 mu m) of selenium deposits indicated that bioreduction occurred in the outer zone of the microbial aggregates. In that outer layer, SEM analysis showed abundant intracellular and extracellular Se-0 (nano)spheres, with some cells having high numbers of intracellular Se-0 spheres. Electron tomography showed that microbial cells can harbor a single large intracellular sphere that stretches the cell body. The Se-0 spheres produced by the microorganisms were capped with organic material. X-ray photoelectron spectroscopy (XPS) analysis of extracted Se-0 spheres, combined with a mathematical approach to analyzing XPS spectra from biological origin, indicated that proteins and lipids were components of the capping material associated to the Se-0 spheres. The most abundant proteins associated to the spheres were identified by proteomic analysis. Most of the proteins or peptide sequences capping the Se-0 spheres were identified as periplasmic outer membrane porins and as the cytoplasmic elongation factor Tu protein, suggesting an intracellular formation of the Se-0 spheres. In view of these and previous findings, a schematic model for the synthesis of Se-0 spheres by the microorganisms inhabiting the granular sludge is proposed.