ALBERT

Description: In the paper "Salinity as a tool for strain selection in recirculating land-based production of Ulva spp. from germlings to adults" (Cardoso et al. 2023) there was a need to compare artificial and natural seawater based on their nutrient composition and concentration. This experiment was performed to guarantee that the results from the other experiments in this paper were not caused by the different types of water, considering that before the start of these experiment the original cultures were growing in natural seawater. The artificial seawater (30 PSU) (Seequasal-Salz, Seequasal Salz Production and Trade GmbH, Münster, Germany) used in this experiment was made in the laboratory at the Alfred Wegener Institute (AWI), Bremerhaven, Germany, right before being analysed. The natural seawater (30 PSU ± 2 PSU) was collected by the Research Vessel Uthörn in the surrounding area of Helgoland, Germany. The process of collection of natural seawater was made every week to refill and supplement the tanks at AWI, Bremehaven. Therefore, the time of the collection and specific location are not known. The nutrient concentration analysis was done in October 2022 at AWI. Before the analysis both water types were pasteurized for 4 hours at 99 °C. The samples collected for analysis were filtered (0.2 µm) (Nalgene®, Nalge Nunc International, USA) and separated into falcon tubes (n = 3). The analysis was performed by an auto-analyser (SEAL Analytical, United Kingdom) and the concentrations of Phosphate, Ammonium, Nitrite and Nitrate were measured.

Description: In the paper "Salinity as a tool for strain selection in recirculating land-based production of Ulva spp. from germlings to adults" (Cardoso et al. 2023) the relative growth rates of germlings of different Ulva species and strains were measured to evaluate the impact of different salinity treatments. We hypothesised that, since their early stages, salinity impacts the relative growth rate of the seaweeds and that different strains are adapted to different salinities. With this data it was possible to use salinity as a tool for selecting the strain with the highest and determine the optimal salinity to grow its germlings under a nursery setting to supplement a large-scale production. Four strains were tested (U. lacinulata and U. linza from the NE-Atlantic and U. lacinulata and U. flexuosa from the Mediterranean). The NE- Atlantic strains were collected in the Óbidos Lagoon, Portugal in January 2021 and were cultivated in laboratory conditions in the Alfred Wegener Institute (AWI), Bremerhaven, Germany. The Mediterranean strains belong to an AWI collection and were isolated in 1986 and 1987 (U. flexuosa and U. lacinulata, respectively). The cultivation of the Mediterranean species with the purpose of using them for experiments started in June 2021. To recreate the conditions of a nursery system, the strains were grown previously and during the experiment in a medium of artificial seawater (Seequasal-Salz, Seequasal Salz Production and Trade GmbH, Münster, Germany) enriched with half-stregth Provasoli (PES; Provasoli 1968; modifications: HEPES-buffer instead of TRIS, double concentration of Na₂glycerophosphate; iodine enrichment following Tatewaki, 1966). Three germlings with similar size from each species and population were placed into multi-well plates and subjected to different salinity treatments (10, 15, 20 and 30 PSU) (n = 3). The germlings were cultivated for 3 weeks. Because of the small size of the germlings, measuring the fresh weight was not possible and, in this experiment, pictures of the germling development were taken every week and the area of the germlings was measured with the software Image J (Rasband 2012). The difference in size was later used to determine their relative growth rate.

Description: In the paper "Salinity as a tool for strain selection in recirculating land-based production of Ulva spp. from germlings to adults" (Cardoso et al. 2023) the antioxidant activity (AA) experiment aimed to test a previously selected strain, Ulva lacinulata from the NE-Atlantic, and evaluate the variations in AA under different salinity treatments. We hypothesized that by reducing the salinity level in the recirculating land-based system, it would be possible to optimize and increase the quality of the biomass being produced before it being harvested. The NE-Atlantic seaweed was collected in the Óbidos Lagoon, Portugal in January 2021 and was cultivated in laboratory conditions in the Alfred Wegener Institute (AWI), Bremerhaven, Germany. To recreate the conditions of a large-scale system, the strain was grown previously and during the experiment in a medium of artificial seawater (Seequasal-Salz, Seequasal Salz Production and Trade GmbH, Münster, Germany) enriched with a cheaper commercial fertilizer Blaukorn (COMPO SANA®, Germany). Twelve 1 L beakers were filled with artificial seawater at 4 different salinities (10, 15, 20 and 30 PSU), measured with a refractometer (Atago, Japan) (n = 3). Six discs (2 cm) of U. lacinulata were placed in each beaker and, at different times (0 h, 3 h, 24 h, 120 h, 192 h and 240 h) 1 disc was collected from each beaker to evaluate the antioxidant activity. The AA was determined by the ABTS Radical Cation Decolourisation Assay based on Re et al., 1999, and a Trolox standard curve was created by measuring the absorption of different Trolox concentrations in ethanol after being mixed with ABTS. The absorption of the samples at 734 nm was analysed with a microplate reader (Infinite 200 Microplate Reader, Tecan Trading AG, Männedorf, Switzerland) and the AA results obtained were given in Trolox Equivalent (in µg/mL).

Description: In the paper "Salinity as a tool for strain selection in recirculating land-based production of Ulva spp. from germlings to adults" (Cardoso et al. 2023) it was evaluated the impact of different nutrient sources on the relative growth rates (RELATIVE GROWTH RATE) of four different Ulva strains during their adult stage. Previously to this experiment, two experiments were performed to evaluate the of germlings and adults of the four Ulva strains. Because the aim of these experiments was to reproduce the conditions of a large-scale production and a small-scale nursery, the adult material was supplemented with a cheaper commercial fertilizer Blaukorn (COMPO SANA®, Germany) while the germlings (in the nursery set-up) were supplemented with half-strength Provasoli (PES; Provasoli 1968; modifications: HEPES-buffer instead of TRIS, double concentration of Na₂Glycerophosphate; iodine enrichment following Tatewaki, 1966). Therefore, this experiment was performed to guarantee that the use of different nutrient sources would not impact the results of the former experiments. Four strains were tested (U. lacinulata and U. linza from the NE-Atlantic and U. lacinulata and U. flexuosa from the Mediterranean). The NE- Atlantic strains were collected in the Óbidos Lagoon, Portugal in January 2021 and it was cultivated in laboratory conditions in the Alfred Wegener Institute (AWI), Bremerhaven, Germany from that moment on. The Mediterranean strains belong to an AWI collection and were isolated in 1986 and 1987 (U. flexuosa and U. lacinulata, respectively). The cultivation of the Mediterranean species with the purpose of using them for experiments started in June 2021. Fresh thalli from each species and population were placed into 1 L glass beakers and subjected to one of the two treatments: water enriched with commercial fertilizer Blaukorn or with half-strength Provasoli (n = 3). The experiment ran for 3 weeks. The fresh weight was measured once a week by collecting the seaweed and removing the excess water with absorbent paper three times before weighing the samples (Sartorius, Germany). Every time, each sample was measured 3 times. Based on the fresh weight measured each week, it was possible to calculate the of each strain throughout the experiment.

Description: In the paper "Salinity as a tool for strain selection in recirculating land-based production of Ulva spp. from germlings to adults" (Cardoso et al. 2023) the relative growth rates of adult thalli of different Ulva species and strains were measured to evaluate the impact of different salinity treatments. We hypothesised that salinity impacts the of the seaweeds and that different strains are adapted to different salinities. With this data we aimed to use salinity as a tool for selecting the strain with the highest and determine the optimal salinity to grow it in the future in a recirculating land-based system. Four strains were tested (U. lacinulata and U. linza from the NE-Atlantic and U. lacinulata and U. flexuosa from the Mediterranean). The NE- Atlantic strains were collected in the Óbidos Lagoon, Portugal in January 2021 and were cultivated in laboratory conditions in the Alfred Wegener Institute (AWI), Bremerhaven, Germany. The Mediterranean strains belong to an AWI collection and were isolated in 1986 and 1987 (U. flexuosa and U. lacinulata, respectively). The cultivation of the Mediterranean species with the purpose of using them for experiments started in June 2021. To recreate the conditions of a large-scale system, the strains were grown previously and during the experiment in a medium of artificial seawater (Seequasal-Salz, Seequasal Salz Production and Trade GmbH, Münster, Germany) enriched with a cheaper commercial fertilizer Blaukorn (COMPO SANA®, Germany). A uniform amount of fresh thalli from each species and population were placed into 1 L glass beakers with salinities of 10, 15, 20 and 30 PSU (each condition n = 3) and cultivated over 3 weeks. The fresh weight was measured once a week by collecting the seaweed and removing the excess water with absorbent paper three times before weighing the samples (Sartorius, Germany). Every time, each sample was measured 3 times. Based on the fresh weight measured each week, it was possible to calculate the of each strain throughout the experiment.

Description: This study examined the growth response of juvenile turbot (Scophthalmus maximus) to diets with graded fishmeal (FM) replacement with plant, animal, and emerging protein sources (PLANT, PAP, and MIX) in comparison to a commercial-like diet (CTRL). The feeding experiment was carried out from April to July 2019 in the Centre for Aquaculture Research (ZAF) at the Alfred Wegener Institute for Polar and Marine research in Bremerhaven, Germany. The juvenile turbot (Scophthalmus maximus) were purchased from France Turbot (L'Épine, France) and acclimated to the recirculating aquaculture system (RAS) for 2 weeks prior to starting the 16 weeks experimental trial. To elucidate the effects of the protein sources and the level of FM replacement on the proximate and mineral composition of the carcass and the apparent digestibility of the diets, in this study. The chemical analysis of the diets was conducted in duplicates and of the carcass and faeces as pooled replicates per tank (n =5 tanks per diet). The carcass samples were minced frozen using a meat grinder (MADO Primus, Germany), refrozen at−20 °C and then freeze-dried for 48 h. The samples of the experimental diets and faeces were freeze-dried for 24 h. The experimental diets and whole body samples were further homogenised in a knife grinder (5000 rpm, 30 s, Grindomix GM 200, Retsch, Germany). The moisture content, ash, crude protein, crude lipid and energy of the experimental diets, carcass and faeces was determined after AOAC (1980). Moisture content of the feeds was determined by drying the samples at 105 °C for 24 h. The moisture content of the whole body and faeces was determined by freeze-drying. Total ash content was determined by combustion of the samples in a muffle oven at 550 °C for 6 h. The total nitrogen in the feed and whole body samples was determined following the automated Kjeldahl Method. Due to small sample volume in the faeces samples, the total nitrogen was determined after the Dumas method. For all samples, the measured total nitrogen was converted to equivalent crude protein (%) by the numerical factor of 6.25. Crude lipid was determined by acid hydrolysis. Gross energy was measured in an adiabatic bomb calorimeter (Model 6100; Parr Instrument, Germany). For the analysis of the mineral content, 0.2 g of freeze-dried and homogenised samples of the experimental diets, whole body and faeces was digested in 3 mL nitric acid (HNO3) (65%, trace grade) in a microwave oven (CEM MARS5, Germany) according to DIN EN 13,805 (2014). After digestion, the samples were diluted with Milli-Q water to 50 mL. Calcium, potassium, magnesium, phosphorus, arsenic, copper, iron, manganese, yttrium and zinc concentrations were analysed in an ICP-OES (iCAP7400; Fisher Scientific, Germany). As reference fish muscle (ERM – BB422, EU) was used.

Description: This study examined the growth response of juvenile turbot (Scophthalmus maximus) to diets with graded fishmeal (FM) replacement with plant, animal, and emerging protein sources (PLANT, PAP, and MIX) in comparison to a commercial-like diet (CTRL). The feeding experiment was carried out from April 2019 to July 2019 in the Centre for Aquaculture Research (ZAF) at the Alfred Wegener Institute for Polar and Marine research in Bremerhaven, Germany. The juvenile turbot (Scophthalmus maximus) were purchased from France Turbot (L'Épine, France) and acclimated to the recirculating aquaculture system (RAS) for 2 weeks prior to starting the 16 weeks experimental trial. To elucidate the effects of the protein sources and the level of FM replacement on the feed performance, in this study, a diet with a balanced mixture and higher level of FM replacement was included. The fish were fed twice a day (9 am and 2 pm) ad libitum. After the fish were fed in the afternoon (30 min later), the remaining pellets were netted (mesh size 1 mm) from the tanks, dried for 24 h at 50 -°C and weighed. To account for potential weight loss of the non-eaten pellets, duplicates of each experimental diet (2 g each) were incubated at 16 -°C and 100 cycles per minute in 100 mL water which was taken from the experimental recirculation system (30 °% salinity) (Obaldo et al. 2002).

Description: This study examined the growth response of juvenile turbot (Scophthalmus maximus) to diets with graded fishmeal (FM) replacement with plant, animal, and emerging protein sources (PLANT, PAP, and MIX) in comparison to a commercial-like diet (CTRL). The feeding experiment was carried out from April 2019 to July 2019 in the Centre for Aquaculture Research (ZAF) at the Alfred Wegener Institute for Polar and Marine research in Bremerhaven, Germany. The juvenile turbot (Scophthalmus maximus) were purchased from France Turbot (L'Épine, France) and acclimated to the recirculating aquaculture system (RAS) for 2 weeks prior to starting the 16 weeks experimental trial. To elucidate the effects of the protein sources and the level of FM replacement on the growth performance, in this study, a diet with a balanced mixture and higher level of FM replacement was included. Fish were weighed to 0.2 g precision and measured in length to 0.5 cm precision every 4 weeks.

Description: This study examined the metabolic response of juvenile turbot (Scophthalmus maximus) to diets with graded fishmeal (FM) replacement with plant, animal, and emerging protein sources (PLANT, PAP, and MIX) in comparison to a commercial-like diet (CTRL). The feeding experiment was carried out from April to July 2019 in the Centre for Aquaculture Research (ZAF) at the Alfred Wegener Institute for Polar and Marine research in Bremerhaven, Germany. The juvenile turbot (Scophthalmus maximus) were purchased from France Turbot (L'Épine, France) and acclimated to the recirculating aquaculture system (RAS) for 2 weeks prior to starting the 16 weeks experimental trial. To elucidate the effects of the protein sources and the level of FM replacement on the metabolic response of the fish, a 1H‐nuclear magnetic resonance (NMR) spectroscopy was used to assess the metabolic profiles of muscle and liver tissue after feeding the fish the experimental diets for 16 weeks. Feed, muscle, and liver samples were ground under liquid nitrogen and approx. 200–250 mg tissue was homogenized in 5x volume of ice‐cold 0.6 M perchloric acid (PCA) (w:v). After one cycle of 20 s at 6000 rpm and 3 °C, using Precellys 24 (Bertin Technologies, Montigny‐le‐Bretonneux, France), samples were sonicated for 2 min at 0 °C and 360 W (Branson Sonifier 450, FisherScientific, Schwerte, Germany). Homogenates of the experimental diets, muscle and liver tissues were centrifuged for 2 min at 0 °C and 16,000 g, and supernatants were neutralized with ice cold potassium hydroxide (KOH) and PCA to pH 7.0–7.5. To remove precipitated potassium, perchlorate samples were centrifuged again for 2 min at 0 °C and 16,000 g. The entire supernatant was transferred, shock‐ frozen in liquid nitrogen, and stored an −80 °C for later analysis. One‐dimensional 1H‐NMR spectra for feed and tissues extracts were acquired using a vertical 9.4 T wide bore magnet with Avance III HD (Bruker‐GmbH, Ettlingen, Germany) at 400.13 MHz with a 1.7 mm diameter triple tuned (1H‐13C‐15N) probe. Each spectrum was processed and analyzed with Chenomx NMR Suite 8.4 software (Chenomx Inc., Edmonton, Canada). Before analyzing, the spectra were corrected for phase, shim and baseline and calibrated to trymethylsilyl proprionate (TSP) signal (at 0.0 ppm).