Qualitative and quantitative diversity of microorganisms present in anaerobic granular sludges fed with different substrates, as well as the structure of these granules have been studied using fluorescent 16S rRNA-targeted in situ hybridization and electron microscopy. The granules showed a multi-layered structure, in which both densely packed and loose micro-colonies, channels and holes could be observed. Only bacteria were found in the outer shell of the granules, while both archaea and bacteria were detected in the inner core. Although high cell density was found in the granules (more than 1011 cells/gram, determined by DAPI-stain) only a low percentage of cells was able to hybridize with the rRNA-targeted probes. Significant quantitative and qualitative differences were observed in the composition of granules fed with different substrates (formate, acetate at high and low concentrations, propionate, sucrose, starch and peptone). Bacterial cells were mostly gram-positives. Active proteobacteria were scarce in the granules exposed to VFA. Syntrophobacteria became dominant in the propionate-grown biomass. Concerning methanogenic archaea, Methanosaeta was the predominant species using complex substrates or low acetate concentration fed granules, while Methanosarcina and members of Methanobacteriales were predominant in the granules grown at high concentration of acetate or formate, respectively. Other Methanomicrobiales and Methanococcales, have been detected in the anaerobic granular sludge in the conditions used in this work.

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