The germination of Gelidium floridanum tetraspores was not stimulated by adding CaCl2, as there was no significant difference compared to the control. This result differs from Bouvie (2018), where tetraspores treated with 2 mM CaCl2 showed a significantly higher germination rate than the control and germ tube formation in 4 hours of cultivation.
In general, the shell powder caused a lower germination rate than the control. Tetraspores from these treatments showed a change in cell division pattern, with alteration in the migration of cytoplasmic content and successive cell divisions as well as an alteration in the formation of rhizoids. In particular, tetraspores treated with oyster powder started germination but died in the process, indicated by the presence of a greenish mass. This greenish mass probably comes from tetraspores that did not adhere to the substrate, dying and disintegrating (Simioni et al. 2015). Possibly, even after filtration, the shell powder solution presented microparticles deposited on the slides, making it difficult to tetraspores adhesion, causing their degradation.
Moreover, the oyster shell has calcite in its composition, making the powder finer than mussel's powder because of the less organized molecular structure and fragile bonds. This finer powder could also influence the tetraspores' development since there was an apparent difference between them and those cultivated in mussel powder. For instance, after 20 cultivation days, germlings cultivated in Ca2 showed dense clusters, while those from shell powder showed small low-density clusters. In particular, germlings from Os1, Mx1, and Mx2 were shorter than other treatments and control. Probably, these results are a consequence of what happened in the germination process, confirming the adhesion of tetraspores cultivated in shell powder.
The explants from Mx1 and Mx2 developed upright axes in both ends with significantly higher numbers and growth rates. On the other hand, explants treated with CaCl2 showed upright axes in only one extremity. The calcium gradient in the cytoplasm is responsible for F-actin organization, which results in the correct cell polarization (Hable and Hart, 2010). The higher calcium concentration CaCl2 treatment (78.4 mg L− 1) is slightly higher than the lowest mussel powder (68 mg L− 1) treatment. In this case, it seems that calcium availability is not the only factor influencing the polarization. Oyster powder treatments, in turn, resulted in the degradation of the thallus. This degradation probably is not caused by calcium since the concentration (84 and 133 mg L− 1) is slightly higher than mussel powder. Since the main component of the powder is calcium carbonate, the toxicity of carbon dioxide should be considered. Hamester et al. (2012) characterized the calcium carbonate of the same species used in the present work, finding that there are no significant differences in the percentage of calcium oxide (CaO) and sulfur trioxide (SO3); however, these oxides were the only common, and mussels powder showed more diversity in other oxides, including K2O, SiO2, SrO, Fe2O3, MgO and Al2O3. Probably, these cations are working as a buffer in the seawater, avoiding the toxicity caused by the carbonate excess. More detailed studies are needed to understand the effects of these oxides in the growth of G, floridanum.
Santa Catarina is the leading Brazilian Producer of mollusks (EPAGRI 2019). Thus, there is high consumption of mussels and oysters, generating a high amount of shell waste. These wastes can be purchased free of charge. There is no need to use a chemical process during the transformation into powder, under the conditions described, offering a sustainable solution if it could be used in Gelidium farming, replacing CaCl2.
In conclusion, the shell powder did not improve tetraspores' germination, but it can be considered to cultivate explants since it stimulates new upright axes. Mussel powder at 252 mg L− 1 is recommended rather than oyster powder.