Manipulation experiment prokaryotic gross growth rates
Surface water was collected at the Blanes Bay Microbial Observatory (BBMO), a shallow (~20m depth) costal station located ~1km offshore in the North Western Mediterranean Sea (41º40'N, 2º48'E) at the four astronomical seasons of 2017: winter (February 21st), spring (April 26th), summer (5th July) and fall (November 7th). Seawater was sieved through a 200-um mesh and transported to the laboratory within 2h. The manipulation experiments started the following day of the four samplings. Each experiment had six different treatments, which consisted on reducing cumulative growth rates controlling factors: Control (CT): incubation of untreated seawater, predator reduced (PR): seawater prefiltered through 1-um filter to remove most predators. Both (CT and PR) had two light cycles: natural light/dark cycle (CL and PL), and darkness (CD and PD), respectively. A diluted treatment (DL): whole seawater diluted 1:4 with 0.2-um filtered seawater to reduce both predation and competition for nutrient and organic carbon resources exposed to natural light/dark cycles. A virus-reduced treatment (VL): whole seawater diluted 1:4 with seawater filtered through a 30-kDa VivaFlow (tangential flow filtration) cartridge to reduce predation, viruses and resource competition, also exposed to natural light/dark cycles. The light treatments were limited to photosynthetically active radiation (PAR) by maintaining the bottle incubations under natural light conditions with the exclusion of UV radiation, using two layers of an Ultraphan URUV Farblos Filter and a net that reduced light intensity to roughly mimic the light conditions of 3 m water depth, calculated from the transparency measures at the sampling site. PAR was monitored continuously in the incubation water baths. The dark treatments were completely covered with two layers of black plastic bags to prevent light exposure. The experiments were performed in 9L Nalgene bottles per triplicate, which were incubated in 200L baths with circulating water to maintain temperature close to in situ conditions during 36/48 hours (until cell abundance reached the stationary phase) and sampled every 12 hours approximately. A total of five samples were taken for nucleic acid extraction and cell counts: one right after sampling for characterizing the natural community, at t0 of the incubation experiments, and approximately every 12h (t2, t3, t4).