water samples collected from metalimnion in Ry de Rome in July 2010
Samples for DNA extraction were sequentially passed through 10.0 µm pore–size 47 mm diameter filters (Nuclepore), to remove particulate debris and large planktonic organisms, and then through 0.22 µm pore–size 47 mm diameter DURAPORE® (Millipore, USA) filters to retain free–living prokaryotes. Filters were preserved in eppendorfs containing 2.0 mL Sucrose Buffer (Tris HCl 1 M pH 8.0, EDTA 0.5 M pH 8.0, Sucrose 1.5 M) at -20ºC until further analyses. Total nucleic acids were extracted from 0.22 µm pore–size filters using enzymatic and mechanical cell lysis procedures. DNA concentration and purity were determined using a Nanodrop ND-2000 UV-Vis spectrophotometer (Nanodrop, DE). Finally, nucleic acid extracts were kept at -20ºC until further analyses.  Bacterial tag-encoded 454 FLX amplicon pyrosequencing (b-TEFAP) were performed using a 454 FLX Titanium platform (Roche) with Titanium reagents at Research and Testing Laboratory (Lubbock, TX, USA) as previously described (Dowd et al., 2008; Capone et al., 2011; Lenchi et al., 2013). The universal bacterial primers 28f (5’-gAg TTT gAT CNT ggC TCA g) and 519r (5’-gTN TTA CNg Cgg CKg CTg) were selected to span the V1–V3 variable region of the 16S rRNA gene.