ALBERT

Description: Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.

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: We investigated effects of temperature during ontogeny of the kelp Laminaria digitata across haploid gametophyte and diploid sporophyte life cycle stages in 5 distinct genetic lines. We sampled meristematic discs from wild sporophytes on the island of Helgoland (North Sea; 54.1779 N, 7.8926 E) in May 2017, July 2017 and February 2018 and conducted a seasonal growth experiment at 5°C and 15°C over 14 days (experiment 1). Based on meiospores from five individuals (genetic lines) sampled in July 2017, we applied a full-factorial experimental design to generate different temperature histories by applying 5°C and 15°C during meiospore germination, gametogenesis of parental gametophytes and recruitment of offspring sporophytes (19-26 days; recruitment), and juvenile sporophyte rearing (91-122 days). We then tested for thermal plasticity among temperature history treatments at 5°C and 15°C in a final 12-day experiment assessing growth, the storage compound mannitol, carbon and nitrogen contents, and fluorometric responses in 3-4 month old sporophytes (experiment 2).

Description: In a mechanistic investigation of heat stress, heterosis (hybrid vigour), and underlying gene expression patterns, we assessed the thermal performance of inbred (selfings) and outbred (reciprocal crosses) sporophytes of the N-Atlantic kelp Laminaria digitata among clonal isolates from two divergent populations; one from the temperate North Sea (Helgoland) and one from the Arctic (Spitsbergen). First, we investigated the upper thermal tolerance of microscopic sporophytes in a 14-day experiment applying sublethal to lethal 20–23°C. We then subjected 4–7 cm long sporophytes to a control temperature (10°C), moderate (19°C) and sublethal to lethal heat stress (20.5°C) for 18 days to assess the physiological parameters growth and optimum quantum yield.