The Ulva genus has proved to be a suitable candidate for large-scale cultivation and can serve multiple purposes in several industries (e.g., pharmacy, cosmetics, food, and feed) (Mantri et al. 2020; Leyva-Porras et al. 2021). However, to guarantee the success of its large-scale production, it is important to find solutions to the currently existing biological and technical limitations. Some Ulva species reproduce in predictable ways, and their reproduction can be induced by following established methodologies (see Mantri et al. 2020 for recent review). However, attempts at inducing the reproduction of the commonly cultivated species Ulva lacinulata (Kützing) have been unsuccessful, and large-scale cultivation of this species requires re-stocking from the wild or limiting the harvest to maintain a starting stock of biomass. While this species is often sought for cultivation because it grows well unattached and it does not sexually reproduce, which results in the loss of biomass, this species often degrades, which also contributes to a loss of biomass. The cause of this biomass degradation has until now not been understood. Here, we present evidence that tissue degradation in U. lacinulata occurs as a result of natural protoplast production and release. These protoplasts can then develop in several different directions. New blades can be formed, which either develop into adult thalli or undergo gametogenesis very early in their development, resulting in the release of gametes and the germination of new germlings. These results contribute to a new understanding of the life cycle of U. lacinulata, and provide the first evidence of natural protoplast production in this species. Because the induction of protoplasts in Ulva spp. is time-consuming and expensive, finding the trigger for this process will be an important step to accelerating the success of large-scale cultivation of this species.
The biological material (previously molecularly identified as Ulva lacinulata by Cardoso et al. 2023) was collected in 2021 in a “green tide” area in Lagoa de Óbidos, Portugal (following the Nagoya Protocol) and then brought to the Alfred Wegener Institute, Germany where it has been cultivated in a closed system. Throughout its cultivation, it was kept in 5 L glass vessels at 15 °C (± 1 °C) and an irradiance of 70 μmol photons m−2 s−1 with a 16:8 h light:dark photoperiod (LD) in pasteurized artificial seawater (ASW) (30 PSU) supplemented with half-strength Provasoli in a concentration of 10 mL L-1. The medium was replaced once per week and an aeration system guaranteed the continuous tumbling of the material inside of the vessels.
Upon observation of the onset of biomass degradation, fresh biomass (0.72 g) was collected. The biomass was distributed equally into four 1 L beakers. The experiment ran for four weeks under the previously mentioned cultivation conditions. Every week, the water in each beaker was filtered and centrifuged to collect the protoplasts. After filtering the water, the debris and small pieces of the original biomass were collected, weighed, and placed back into the beaker with newly added culture media. Calcofluor white (CFW) was used to observe the presence/absence of cell walls and disposable hemacytometers were used to quantify the protoplasts yields obtained in each beaker each week. A defined amount of the collected protoplasts were isolated in individual Petri dishes and their development was observed under the microscope. The number of protoplasts that germinated into new blades were counted after five weeks.
Results and Discussion
Our observations under the fluorescent microscope confirmed the absence of cell walls in the cells dyed with CFW, thus confirming that these cells are naturally occurring protoplasts. The total protoplast yield obtained in our first experiment was 3.21 x 107 cells per gram of fresh biomass, which is comparable to those reported in studies where the protoplast formation of different Ulva species was enzymatically induced (Reddy et al. 2018). Approximately half of the protoplasts regenerated (40-60 %) and grew into discs or germlings in a similar fashion to what has been described in the literature for induced protoplasts of Ulva spp (Reddy et al. 2018; Fig. 1). Moreover, sexual reproduction was found to occur during gametogenesis in protoplasts, rather than in adult blades.
Additionally, by measuring the weight of the initial biomass each week, we observed that the original tissue did not completely degrade after protoplast formation and release, and a final total fresh weight of 5.79 g resulted in a 7 % daily relative growth rate. Thus, revealing that degradation does not necessarily result in a total loss of the culture.
Our observations of natural protoplast production in U. lacinulata close an important knowledge gap in understanding this species’ reproductive cycle. This new knowledge can be beneficial when trying to understand the formation of “green tides” and the differences between “green tide” strains and non-“green-tide” strains. Additionally, the production of natural protoplasts can potentially be exploited to improve the efficiency of Ulva cultivation methods in the future and remove the bottlenecks existing today for safe and profitable large-scale production.
Cardoso I, Meissner A, Sawicki A, Bartsch I, Valentin K-U, Steinhagen S, Buck BH & Hofmann LC (2023) Salinity as a tool for strain selection in recirculating land-based production of Ulva spp. from germlings to adults. J Appl Phycol (Accepted, under review).
Leyva-Porras C, Román-Aguirre M, Cruz-Alcantar P, Pérez-Urizar JT & Saavedra-Leos MZ (2021) Application of Antioxidants as an Alternative Improving of Shelf Life in Foods. Polysaccharides, 2(3): 594–607. https://doi.org/10.3390/polysaccharides2030036.
Mantri VA, Kazi MA, Balar NB, Gupta V & Gajaria T (2020) Concise review of green algal genus Ulva Linnaeus. J Appl Phycol 32(5): 2725–2741. https://doi.org/10.1007/ s10811-020-02148-7.
C RK & Seth A (2018) Manual for protoplast preparation from Ulva and their seeding for cultivation. Biowaste and algae knowledge for the production of the 2nd generation biofuels: BioWALK4Biofuels. CSIR- CSIR-Central Salt and Marine Chemicals Research Institute. https://doi.org/10.13140/RG.2.2.20466.15048.