2 N HCl and rinsed three times with Milli-Q and filtered lake water. All the bottles were incubated in the lake at 2 m depth for four complete days. Subsamples were taken from each triplicate at day 0, 2 and 4 to assess microbial abundances and activities, and at day 0 and 4 for the analysis of the bacterial community diversity. Flow cytometry (FCM) Selleck AZD5582 sample analysis We used a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, U.S.A.)

equipped with an air-cooled laser providing 15 mW at 488 nm with the standard filter set-up. Only a few hours after sampling (less than 4 h), one millilitre of water was immediately analysed without adding any fixative or dye to analyse the picocyanobacterial community dynamics and also to check for the absence/presence of prokaryotic (e.g. Synechococcus) and eukaryotic autotrophic organisms in the V treatment. Such unfixed samples, kept in darkness in refrigerated boxes and at 4°C for a few hours before analysis, revealed no significant changes in cell counts while this was not true when using either formaldehyde or glutaraldehyde (not shown). Fluorescent microbeads (Molecular Probes Inc., Eugene, OR, U.S.A.) of 1-μm diameter were added to each sample as an internal standard. Another 1 ml was fixed and used for bacterial and viral counts via FCM, according to

the protocol described in Personnic et al. [25]. Briefly, viruses were fixed with glutaraldehyde (0.5% final concentration, grade I, Merck) for 30 min in the dark, then diluted Glycogen branching enzyme in 0.02 μm filtered TE buffer (0.1 mM Tris-HCL and 1 mM EDTA, pH 8), and incubated with SYBR Green I (at a final 5 × 10-5

4EGI-1 dilution of the commercial stock solution; Molecular Probes), for 5 min at ambient temperature, followed by 10 min at 75°C, and then another 5 min at room temperature, prior to FCM analysis. Heterotrophic bacterial counts were performed on samples that had also been fixed with glutaraldehyde (0.5% final concentration) for 30 minutes, but the samples were then diluted in 0.02 μm filtered deep-lake water sample, and incubated with SYBR Green I (10-4 dilution of the commercial stock solution) for 15 min [25] Listmode files were analysed using Cytowin [58]. Enumeration of flagellates 50 ml sub-samples were fixed with glutaraldehyde (1% final concentration), stained with primuline [59] and collected onto black polycarbonate membranes (0.8-μm pore size). For flagellates, slides were prepared within 24 h after sampling and were stored at -25°C in darkness to minimise the loss of autofluorescence [60]. Slides were observed at a 1,250× magnification using an epifluorescence microscope (Nikon Eclipse TE200) under UV light for heterotrophic nanoflagellates and, under blue and green light for pigmented nanoflagellates. Bacterial production The incorporation of 3H-leucine was determined following the protocol of Kirchman [61]. For each sample, 5 sterile eppendorfs (2 ml) received 1 ml of sub-sample. Two samples were fixed with formaldehyde (1.