Physicochemical and biological characterization of long-term operated sulfate reducing granular sludge in the SANI® process. 
The SANI(®) process (Sulfate reduction, Autotrophic denitrification and Nitrification Integrated) is a treatment system with low energy demands.  The major bioreactor of this new technology is a sulfate-reducing up-flow sludge bed (SRUSB) that converts organics and provides electron donors for subsequent autotrophic denitrification.  This research characterizes the granules inside the SRUSB, with the aim of improving its efficiency, maximizing its operational flexibility, and minimizing its footprint.  The unique sulfate-reducing bacteria (SRB) granules serving in the SRUSB were found to increase the resilience and compactness of the SRUSB.  The granules, with a compact and porous structure, showed high cohesion resisting breakage with a shear force G > 3400 s(-1).  The hydrophobicity of the external surface of the mature granules remained stable at around 70% and acid volatile sulfide (AVS) accumulated at the bottom of the SRUSB.  16s rRNA gene analysis of the microbial communities revealed that Desulfobulbus (42.1%), Prosthecochloris (19%) and Trichococcus (12%) dominated the mature granular sludge.  Fluorescence in situ hybridization (FISH) further showed that SRB organisms were located internally and then surrounded by non-SRB.  According to the FISH results, the spatial distribution of extracellular polymeric substances (EPS) displayed protein and α-polysaccharides in the exterior and β-polysaccharide in the core of the granules.  Such biological structure suggests that each SRB granule acts as an efficient and independent unit, capable of achieving both fermentation and organic conversion.  The present investigation sheds light on the physicochemical and biological characteristics of the SRB granulate.  This information provides valuable information for scaling-up the SANI(®) process to treat real saline sewage in Hong Kong.