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The remaining 23 mL of each culture was quickly filtered under vacuum (Air Cadet vacuum/pressure station, Thermo), immediately washed with cold saline solution (9.5 g L 1 NaCl at 12 C) and quenched in cold methanol/water (1:1 v/v) at 30 C as described by Smart et al

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Bot activity and speeders were identified, with minimal exclusions