Series 3: Skeletonema marinoi growth and aggregation under future ocean acidification conditions
- Phase 2: Aggregation Phase - After incubation (40hours)
- Experiments 1 and 2

METHODS
2.1 General set up: The experiment consisted of two treatments (Present and Future) representing different pCO2 conditions and two sequential experimental phases: the cell growth phase and the aggregation phase. The experiment was sequentially replicated. The two replicates are going to be referred as Experiment 1 and Experiment 2.

2.2 Cell growth phase: During the cell growth phase, the effect of OA was tested on a culture of S. marinoi that was incubated for five days, in 5 l transparent bags. Two replicate bags were used for each treatment. The pH was measured daily immediately after collection in each bag, while the TA samples were collected for later analysis in one sample per treatment at the beginning and at the end of the cell growth phase. The algal cells were counted, sized and their instantaneous in vivo chlorophyll fluorescence (Ft) and quantum yield (Qy) were measured daily in the morning. The concentration of bacteria and TEP were measured at t 0 ,t2.8 and t4.6. The nutrients concentration was sampled at t2.8 and t4.6. The dry weight, particulate carbon, hydrogen and nitrogen were measured at t4.6. After 5 days the cultures in the two bags per treatment were pooled, the carbonate system was readjusted to the starting conditions and subsequently they were inoculated into cylindrical rolling tanks.

2.3 Aggregation phase: The aggregation phase consisted in the incubation of the cultures into cylindrical tanks on rolling tables (Edmondson, 1989). A total of 8 tanks (4 replicates per treatment) were incubated over the rolling table for two days in the darkness in the environmental room at 15°C. Solid body rotation is established in these rolling tanks within less than three hours (Ploug, Terbrüggen, Kaufmann, Wolf- Gladrow, & Passow, 2010) and the sinking of particles through the water column was simulated and aggregation promoted. After 38 hours of incubation the aggregates sinking velocity, number and size were measured. The algal cell number, bacterial cell number, TEP, dry weight, particulate carbon, hydrogen and nitrogen were evaluated, both for the aggregated fraction and for the surrounding water. The carbonate system was characterized by measuring pH in all the tanks and TA in one tank per treatment.