Biofilter-release
To determine the impact of biofilter biofilm and the bioreactor's dilution rate on the number of planktonic bacteria in the water leaving the reactor, a 56-day experiment was carried out using reactors maintained at temperatures of 12°C and 20°C. The reactors were continuous with 300 mL of seawater and matured AnoxKaldnes K1biofilm carriers (total effective surface area of 662 cm²). The carriers were sourced from a biofilter at NTNU Centre for Fisheries and Aquaculture, Trondheim, Norway. A control reactor without biofilm carriers was operated for each temperature. All reactors were supplied with Seawater M65 medium with an initial flow rate of 12.5 mL/hour (HRT of 24 hours). The dilution rate was gradually increased: an initial rate of 1 d?¹ for 15 days, 0.47 d?¹ for 21 days, 0.21 d?¹ for 14 days, and finally 0.1 d?¹ for 6 days. To determine the release of bacteria from the carriers, a four hours' “release experiment” was carried out. Carriers corresponding to a surface area of 588 cm2 from the 56-days' experiment described above, were transferred to particle-free seawater in separate bioreactors at the relevant temperature. Water samples were collected at intervals: first prior to carrier introduction, then immediately after carrier addition after, and subsequently at 0.5-hour intervals for the next 4 hours. A second experiment with similar design was performed, but instead of two temperatures, two doses of nutrients in the M65 medium was used. For the low nutrient treatment, a 18 µL of M65 medium was added to the reactor (5 L water, 1 L carriers) three days a week for 106 days. For the high nutrient treatment, a dose of 55 µL of M65 medium was added to the reactor (5 L water, 1 L carriers) three days a week for 84 days. The original concentration of the M65 medium stock used was 150 g/L. The release of particles were performed as described above; by transferring carriers from the bioreactors to new reactors containing sterile, particle-free water. Water samples were collected by filtering around 50-60 ml water through 0.2 µm sterile syringe filtered. For biofilm samples, carriers were collected. All samples were kept at -80°C. DNA was extracted using the Power Soil DNA Isolation Kit (MOBIO) as described by the manufacturers. Variable regions 3 and 4 of the bacterial 16S rRNA gene was amplified by using the primers Ill-338F (5-cctacgggwggcagcag) and Ill-805R (5-gactacnvgggtatctaakcc). The amplicon library was sequenced at the Norwegian Sequencing Center in one MiSeq run using v3 reagents and paired end sequencing.