Strain selection and optimization of Ulva spp. for land-based recirculating cultivation systems

Abstract

Ulva species are intertidal macroalgae spread worldwide, known for their biochemical composition (e.g., ulvan) that grants them interesting properties to explore in sectors such as food, feed, cosmetics, pharmaceutical, and bioplastics. They are known for their high growth rates, bioremediation capacity, and plasticity, which can be associated with the formation of harmful green tides. Nevertheless, Ulva is considered a good candidate to cultivate on a large scale. Recirculating aquaculture systems (RAS) are well established systems for the cultivation of fish, shrimp, and other aquatic species and can be operated as a partly flow-through or even complete closed system. Thus, allowing the cultivation of non-native species in land-based recirculating systems without introducingthese species in the wild. Land-based systems present high costs and Ulva cultivation still presents several bottlenecks that limit the possibility of a profitable scale-up process (e.g., variation in biochemical composition, different adjustments, and responses to the environment). The works presented in this dissertation aimed to select Ulva strains for later cultivation in a RAS with artificial seawater and optimize them for their later use as the main constituent of food packaging. Four strains of Ulva from two geographical origins (Ulva flexuosa, Ulva lacinulata, from the Mediterranean Sea, and U. lacinulata and Ulva linza from the NE-Atlantic Ocean) were tested and compared for their robustness and capacity to grow in high temperatures and low salinities. To test the importance of strain selection for Ulva cultivation, eco-physiological experiments were conducted. The relative growth rates (RGR) of different Ulva species and strains under different salinity (Publication I) and temperature conditions were tested (Chapter 6). Strains that presented RGRs below a 7 % day-1 RGR threshold, were considered not ideal. Ulva linza and both strains of U. lacinulata presented elevated growth rates in the temperature range tested. In the salinity experiment (Publication I) it was estimated that RGRs of the adult U. lacinulata strains would be above the 7 % day-1 threshold, even if salinity was reduced to 12 PSU. For this reason, U. lacinulata strains, particularly the NE-Atlantic strain, were considered potential candidates for vegetative cultivation in a RAS under lower salinity settings. To test the same salinity treatments as a potential method for strain optimization, the antioxidant activity (AA) from the NE-Atlantic U. lacinulata was measured for 10 days. AA showed a tendency to increase in the lowest and highest treatments (10 and 30 PSU), suggesting that biomass quality can be increased by further reducing the salinity for a short period before harvesting. Not all the species tested were good candidates for the system, thus corroborating the importance of strain selection before cultivation. Cultivating U. lacinulata strains at lower salinities could amount to a reduction in the costs for the system and an increase in biomass quality. High-quality biomass can increase the profitability of the system by enabling the biomass to be sold for a higher price. Antioxidant activity was considered a necessary property in the strains, as high levels of antioxidants can increase food’s shelf-life. The effect of irradiance on the AA of the two U. lacinulata strains was evaluated (Publication II). Both strains were grown under a saturating irradiance treatment for 5 days and their AA and photosynthetic efficiency were evaluated. Both strains showed the capacity to adapt to the saturating irradiance, but only the NE-Atlantic strain had a significant increase in AA under the saturating irradiance treatment. Thus, suggesting that a significant light increase can improve the quality of the biomass. The lack of differences found in the AA of the Mediterranean strain between the control group and the saturating treatment indicates that light dose (kept the same between the treatments) also plays a role in determining the AA of a strain. The different results from both strains corroborate once more the importance of strain selection and optimization. The NE-Atlantic Ulva lacinulata, often and spontaneously degraded without signs of fertility, and attempts at inducing reproduction were unsuccessful. As a first attempt to overcome these limitations, a successful protoplast isolation method was developed based on the methods reported in the literature. Two eco-physiological pre-treatments (light and salinity) were tested to be used before the isolation of protoplasts to reduce the costs of the isolation method. Only the 40 PSU pre-treatment reduced the cell wall of Ulva-1 (Chapter 6). To understand the constant biomass loss, the degradation process of the NE-Atlantic U. lacinulata was followed (Publication III). The degradation caused the removal of the cell wall and the release of protoplasts into the water that were able to regenerate and regrow. Protoplasts grew into three morphologies (cell masses, unattached discs, and unattached germlings), two of which very soon after regeneration and germination, released gametes into the water. A similar experiment was performed with wild material from Ulva compressa. This species became fertile and released swarmers, but a portion of the cells released were found to be protoplasts. These findings suggest that Ulva species have an unexplored asexual reproduction strategy. This is the first time that the natural formation of protoplast has been reported in Ulva species as a method of reproduction. This knowledge can help us understand Ulva species and how to deal with future degradation events that limit Ulva cultivation.