The experimental site and sample collection: The experimental site, located 15 km from Venice, Italy, has been deeply described in Gumiero et al. (2011). The experimental design was planned according to the NICOLAS project (EC DGXII, 1997–2000 ENV4-CT97-0395) to examine a three-zone buffer system. The particular structure of the experimental field, which is characterized by ridges and furrows, facilitates surface and sub-surface flows of water through the woodland strips. The water drains through the field from the irrigation ditch (located on a small ridge) towards the parallel network of drainage ditches as described in details in Gumiero et al. (2011). Water samples were collected on March (M), April (A) and July (J) from three different sites: the river (R), the irrigation ditch or Input water (I) and the output water of the drainage ditch (O). All the samples were collected and analysed in triplicate. For 5-cyano-2,3-di-4-tolyl-tetrazolium chloride (CTC) assay, direct viable count, and plate count analysis, tenfold water serial dilutions were prepared as described by Toffanin et al. (2000). Water chemical parameters Water samples were filtered through a 0.45 µm polyvinylidene fluoride filter (Merck Millipore) and analysed within 24-36 h for dissolved total nitrogen by the persulphate oxidation method (Valderrama, 1981). Principal component analysis (PCA) was performed by the XLSTAT 2007 software to determine the possible relationships among the two seasons and the three sites of sampling in terms of nitrogen (N) removal (mg mL–1). Microscope-based analyses CTC reduction assay was performed using the procedure described in Basaglia et al. (2007): in short, aliquots of water serial dilution (1:10) were incubated with 4 mM CTC in phosphate buffer solution and after 2 h staining, filtered through a 0.2 m pore size black polycarbonate membrane filter (Merck Millipore, Billerica, MA, USA). Filters were air dried and mounted with a low-fluorescence immersion oil (Molecular Probes Inc., Eugene, OR, USA) on glass microscope slides. Viable bacteria were tested using the Live/Dead BacLight bacterial viability kit (Molecular Probes) following the instruction of the manufacturer; after 15 min of incubation, the samples were filtered through a 0.2-mm black polycarbonate filter (Millipore), and the filter mounted as described above. CTC-reduction and viability were observed by fluorescence microscopy (Olympus fluorescent microscope BX60) equipped with a blue 420-nm exciter filter (Olympus BP 490). At least 20 fields or 300 cells were recorded for each sample. Culturable microorganisms Culturable bacteria in water samples were evaluated as described by Rahman et al. (2014): 0.1 mL aliquots of water serial dilution (1:10) were dropped and spread onto the solid media plate count agar. Petridishes were incubated aerobically at 30°C. After 14 days the number and the morphology (shape, size, colour, etc.) of colonies were recorded. Representative colonies of different morphologies were isolated and stored in glycerol at –20° for molecular analyses. DNA extraction from isolated colonies DNA was extracted from single colony by alkaline lysis: one colony was suspended in an Eppendorf tube with 50 µL of lysis buffer (2.5 mL 10% sodium dodecyl sulfate, 5 mL 1 M NaOH, 92.5 mL MilliQ water). After 15 min the suspension was heated at 95°C, then the tube was centrifuged for 5 min at 13,000xg, the supernatant was transferred to a new tube and 90 µL MilliQ water was added. Extracted DNA was stored at –20°C for further molecular analyses. Amplification and sequencing of 16S rDNA from culturable bacteria and diversity index 16S rDNA amplification by polymerase chain reaction (PCR) and sequencing was performed as described in Rahman et al. (2014). The amplified DNA was visualized by gel electrophoresis and sequenced. The most similar bacterial species were found in the GenBank by using BLAST (Basic Local Alignment Search Tool) search (http://www.ncbi. nlm.nih.gov). Diversity was measured by Shannon diversity index (H) (Shannon and Weaver, 1963; Kapley et al., 2007; Ibekwe et al., 2010). Total DNA extraction directly from water Two hundred fifty mL water was filtered through polyethersulfone filters (0.2 µm pore size; Pall Corp., Port Washington, NY, USA). One filter and a half was used to extract DNA using the Power Soil TM DNA Isolation Kit (Mo Bio Laboratories Inc., Carlsbad, CA, USA). DNA isolation was performed according to the manufacturers’ instructions, modified as follows: extra glass beads (0.15-0.30 g, bead size 0.1 mm) were added to the half filter and the cells were disrupted by bead beating (mini-bead beater™; BioSpec products, Bartlesville, OK, USA). Final purification of the extracted DNA was performed using the Wizard® DNA clean-up system (Promega, Madison, WI, USA). This purified DNA was used for PCR Amplification of 16S rDNA as described above.