0.45 mm pore size filters

Water samples were collected once every month between January and November 2011 at reference stations of Lakes Annecy (Lat N 45.8727, Long E 6.1645333) and Bourget (Lat N 45.7469, Long E 5.86015), corresponding to the deepest part of each lake. We obtained 14 samples for Lake Annecy and 18 for Lake Bourget. Twenty-one liters, integrating the water column from surface to 20 m depth, was collected using an electric pump and tubing, and water was stored in a polycarbonate flask placed in the dark at 4 C before filtration steps. A few hours following sampling, 20 l samples were first filtered through a 60 mm mesh and then filtered through 1 mm pore-size filters (Millipore, Bedford, MA, USA). The filtrate (i.e. <1 mm fraction) was concentrated to a volume of 200e250 ml using a 30,000 Da-molecular-weight-cutoff spiral-wound millipore ultrafiltration cartridge (regenerated cellulose, PLTK Prep/scale TFF, 1 ft2; Millipore). To ensure that all remaining small free-living bacteria were removed, we twice filtered the <1 mm concentrated fraction through 0.45 mm pore-size filters (Millipore). The absence of cellular contamination was verified using flow cytometry (not shown).