Infectious diseases are a common and on-going problem for reared aquatic species. Microbial management tools allow for increased control in aquaculture production systems, reducing the spread of pathogenic microbes while improving the health of cultured organisms. Recirculating aquaculture systems (RAS) are one such tool that is used to select for neutral and beneficial microbes that competitively exclude specific and opportunistic pathogens. However, the high investment costs and complexity of RAS inhibits its widespread implementation. Integrated multi-trophic aquaculture (IMTA) holds the potential to reduce or replace some of the expensive and sophisticated units that comprise RAS, making this technology more widely available while contributing value-added product, a proposition that may contribute to global food sovereignty. This project focuse s on the co-culture of three species from different trophic levels in a closed cold-water multi-trophic marine RAS: nori (Porphyra umbilicalis) , Pacific oyster (Crassostrea gigas) , and North Sea prawns (Palaemon sp.). T he effect of different types of nutrient bioremediation ( traditional bacterial biofiltration versus macroalgae) on nutrient uptake efficiency, the microbiome, and the growth and survival of all trophic levels is evaluated.
Materials and Methods
P. umbilicalis was obtained from the Ghent University Phycology Research Group , C. gigas was obtained as T6 spat from Aquacultuur Oostende bvba (Ostend, Belgium) , and Palaemon post-larvae and adults were wild-caught via dip nets from rocky tidal pools in coastal Belgium and Northern France. T hree closed RAS comprised the experimental set-up and were operated at the Laboratory of Aquaculture and Artemia Reference Centre (ARC), Ghent University. Each up-flow RAS had a total fill volume of 500L , composed of three cylindroconical poly-ethylene culture tanks (100L; 90L fill volume) arranged in parallel with outflow to a drum filter ( 40 µm-mesh size ) and protein skimmer before reaching the nutrient bioremediation tank (Figure 1). All systems were filled with filtered natural seawater, operated at a flow rate of 70L /h, a 12:12 L:D light regime, and with an ambient room temperature of 18°C. Three approaches to n utrient bioremediation were compared: a traditional biofilter (system 1) , a traditional biofilter in combination with P. umbilicalis (system 2) and P. umbilicalis alone (system 3) . The biofilter volume was dimensioned to treat the expected nutrient release from the C. gigas and Palaemon sp. as well as the residual nutrients in the microalgae supernatant fed to the C. gigas . P. umbilicalis were stocked based on the volume of the bioremediation tank and optimization for light exposure.
In each cylindroconical tank, a flat base was placed mid-way to provide a horizontal surface area for the Paelaemon prawns. T wo cylindrical PVC basket s (80 mm height and 300 mm diameter) with a 2 mm mesh bottom were stacked in the tank and elevated with Bio-Net blocks in which aeration was fixed . The Bio-Net support consisted of small crevices, providing shelter places for the prawns. Each tank was stocked with 25g (0.28g/L) of Palaemon sp. and 115g (1.28g/L) of C. gigas , the oyster biomass being distributed equally over the two baskets in each tank. All culture tanks received 2% of oyster wet weight in microalgae dry weight in a 1:1:1 ratio of Tetraselmis chuii , Chaetoceros muelleri and Isochrysis galbana , All microalgae were obtained from Proviron (Hemiksem, Belgium). Palaemon prawns are fed with 1.5-2 mm shrimp pellets .
The t otal biomass of each trophic level wa s kept constant in all systems by removing the excess biomass produced over the course of a week. Microbial samples were taken weekly from each nutrient bioremediation tank. Water quality and nutrients (TAN, nitrite, nitrate, phosphate, calcium, and alkalinity ) were measured three times per week in each bioremediation tank prior to the administration of feed . T emperature, pH and dissolved oxygen were assessed daily in each nutrient bioremediation tank and all culture tanks. The wet we ight of all trophic levels was recorded weekly , as well as the dry weight and ash weight of P. umbilicalis and C. gigas , and the shell length and width of C. gigas .
This experiment represents pioneer research into co-cultivation of multiple trophic levels in a closed RAS. Despite this being the first time P. umbilicalis , C. gigas , and Palaemon sp. have been reared in such a system, there have been no detrimental effects on the health and welfare of the organisms. Furthermore, preliminary results indicate no significant differences in growth for C. gigas and Palaemon sp. between the three systems, and the different forms of nutrient bioremediation tested in this experiment have effectively kept all water quality parameters within a biologically suitable range.
Initial data from this innovative research suggests that conventional recirculating aquaculture farm designs may be effectively modified by replacing the traditional biofilter unit with macroalgae, reducing the upfront costs associated RAS production while contributing value-added biomass.
This study is part of the BlueMarine³.Com project funded by the Flemish government through Flanders Innovation and Entrepreneurship (VLAIO) and is facilitated by the Blue Cluster program.