Improved Palmaria palmata hatchery methods for tetraspore release, even settlement and high seedling survival using strong water agitation and macerated propagules
Introduction
Interest in cultivating of Palmaria palmata Linnaeus O. Kuntze (dulse seaweed) in European countries for human consumption and feed ingredient, has increased as knowledge about its nutritious and potential health benefits is increasingly valued [[1], [2], [3], [4], [5]]. Globally, seaweed cultivation is the fastest growing aquaculture sector and provides a low trophic and nutrient extractive source of biomass [[5], [6], [7]]. Several red algae species have been cultivated at small to larger scales in open water, such as Sarcothalia atropurpurea, Gracilaria lemaneiformis and Pyropia umbilicalis (former Porphyra umbilicalis) to secure a source of raw material for agar and carrageenan extraction as well as food, respectively [[8], [9], [10]]. Those studies used spore inoculation methods and a period of calm water to promote settlement of tetra-, and carpospores on bottom-laid structures, such as mollusks valves or nets. Yet, the exploitation of seaweed in Europe is 99% based on harvest from wild stocks [5]. In Europe, Irish seaweed companies contributed with 99% (<100 t FW) of the annual harvest of P. palmata in 2011 [11]. P. palmata is dried and retailed for high-valued niche products at prices reaching ~245 € per kg dry weight (DW). Besides a high content of essential minerals and rich flavor [1,12,13] and relative high content of crude protein of up to 19.3–35% [4,14], P. palmata has been characterized as promising diet supplement for humans and salmon feed due to the content of bioactive peptides, protein hydrolysates and antioxidants [2,15,16]. As demands for P. palmata increase, focus on cultivation rather than wild harvest of this species is important to avoid diminishment of wild populations and to secure a sustainable crop production [1,17]. Cultivated P. palmata requires a relative high nitrogen availability during growth to maintain the dark-red color due to phycobilliproteins [18], which is an important trademark for the species as commercial product. This have led to the idea of farming P. palmata in nutrient riched areas [7,19,20]. However, in near-coastal waters, like Danish inner waters where nutrient levels may be elevated due to run-off and fish farms, water bodies show great spatial and temporal variation in salinity [21], even down to 15 psu (National monitoring survey data, NOVANA). Despite the potential of cultivating in nutrient-rich semi brackish waters [22], the growth of P. palmata seedlings in low saline waters, as inner Danish waters, is yet to be investigated.
Open-sea cultivation of P. palmata requires a land-based hatchery phase to produce seeded substrate, like twine or textile, using released meiotic tetraspores as seeding material [17,23]. However, as of yet, existing seeding and hatchery methodologies for large-scale cultivation are suboptimal and need further development [24]. The normal hatchery process can be divided in three important steps: spore release, spore attachment and seedling growth (Fig. 1A–D).
Tetrasporophytes release tetraspores during fall and winter from mature fertile tissue (sori) [17,25] (A). Current seeding methodology for P. palmata is based on a gravity principle due to the relative large size (Ø~30 μm) and non-mobility of the tetraspores [26,27]. By this method, fertile sori submerged in inoculations tanks release tetraspores, which settle during few days [17]. The spore release is highly unpredictable as ripeness and rupture of the tetrasporangia varies even within tissue sections [25,28,29]. Furthermore, the sticky nature of P. palmata tetraspores often result in settlement of nested spore aggregates [29] of high density on the substrate or on bottom of the tank [24]. As a result, the spore density on substrates can be highly variable and cause low quality seedling lines [24,30]. To compensate for the low spore use efficiency inherent in this method, a 1:1 areal coverage of sori-to-substrate has been used in the seeding phase (B). However, this impose a high demand of sori [26]. Thus, the ratio between the amount of sori needed to seed substrates and the harvest yield is high, which indicate a low seeding efficiency. Using this method, Werner & Dring (2011) estimated a requirement of 135 kg of fertile plants to seed 70 (1.2 ∗ 3 m) to obtain an extrapolated harvest yield of 1.8 ton FW. After settlement, half of the tetraspores germinate into male gametophytes (seedlings), whereas the other half (female gametophytes) remain microscopic or die off (C) as no fertilization step to initiate the development of new tetrasporophytes is implemented in the normal cultivation practice. Young male seedlings are typically nursed for 1–5 months before deployed at sea (D). After 5–8 months of growth at sea, biomass is ready for harvest in late spring when sea temperature is still within the optimal range for growth (6–17 °C) [14,24].
Agitation mediates a physical disturbance at the water-sporangia interface, which likely enhance sporangia wall rupture [28] and augment the spore release rate. In addition, the risk of contamination increases as fertile tissue remains in the sporulation tank for several days [31]. Thus, agitation of water might be important for a faster spore release rate and seedling survival in batch cultures. Likewise, water agitation might increase evenness of settled spores as it simulates the conditions with moderate exposure and strong currents found in the lower intertidal and sublittoral habitat where P. palmata live. In this habitat, P. palmata is often observed in high densities on stipes of Laminariales [25], suggesting that the stipe surface provides a higher chance for spore settlement, survival and growth. After release, spores settle and attach to kelp stipes mainly as result from higher encounter rate enforced by small-scale water eddies [32,33], though other factors, such as stipes surface roughness and herbivory might also be important [34,35]. The stipes of kelps contain an extracellular alginate matrix to which sticky tetraspores might show preference of settlement attributed to the outer layers of mucilage sheath and the adhesive mucilage vesicles [28,36]. Mimicking an alginate surface on cultivation substrates might enhance the settlement and survival of P. palmata seedlings.
Based on the previous findings and challenges as summarized above, we aimed to investigate a modified strategy (Germinate-macerate-agitate; GMA) to optimize release and handling of P. palmata tetraspores to produce seedlings on cultivation substrate, as shown in Fig. 1(E–H). First step was to test the effect of agitation and spore release duration on spore yield (E). Aggregates of released tetraspores were transferred and germinated in petri dish (F) obtaining a mix of spores and seedlings (propagules). After germination, the aggregates were subjected to maceration as pretreatment to dislodge propagules (G) and used as seeding inoculum in agitated tanks to test the ability of propagules to re-attach on substrates. Three levels of agitation and two types of twine were tested. After settling propagules were denoted ‘seedlings’ (H). Finally, we tested the effect of alginate-coated substrate and the level of water agitation on tetraspore settlement density and salinity effect on seedling growth.
Section snippets
Material & methods
Sterile P. palmata (n = 50) were collected by divers the 23rd of August 2016 near Fyns Hoved, Denmark (55.610895°N 10.594308°E) at 4–5 m depth and water temperature of 15 °C. The salinity of the upper water column at the collection site varies seasonally between 14 and 27 psu (National monitoring survey data, NOVANA) but was not measured at sampling time. The plants were transported to indoor tank facilities and cultured in 400 L aerated filtered seawater (1 μm) at 10 °C and exposed to 12:12-h
Spore release (exp. 1)
During the phase of spore release, water agitation mediated a significant effect (P = 0.0308) on the spore yield, whereas the effect of spore release time was not significant (P = 0.1051). The highest spore yield was found under the conditions of high agitation for 3 h and resulted in a spore yield of 67,906 ± 11,303 spores g FW−1 compared to 17,889 ± 3.652 spores g FW−1 in calm water for 1 h. No interaction between agitation level and spore release time was found (Fig. 2).
Preference of spore settlement and seedling survival (exp. 2)
To evaluate treatment
Discussion
The main objective of this study was to investigate how modified conditions in each hatchery step, e.g. tetraspore release, settlement, and early stage growth can improve seedling production of P. palmata. In a stepwise approach, we have demonstrated a seeding method for P. palmata based on water agitation and maceration of germinated propagules for dispersal and re-attachment to cultivation substrate. The novel findings indicate a potent discoid adhesion capability of the young spores and
Conclusions
To establish commercial P. palmata cultivation, a viable hatchery seedling production is crucial as tetraspores are short in supply, spore usage has currently been suboptimal and seedling density affects harvest yield. Based on this study, we suggest a new versatile strategy to optimize the use of P. palmata tetraspores based on germinated spore aggregates subjected to maceration to produce a solution of adhesive propagules showing the ability of discoid re-attachment on substrate. Besides,
Declaration of interest
None.
Acknowledgements
We thank local scuba divers for collecting the P. palmata biomass and Kasper Lenda Andersen for tank maintenance in the hatchery. The study was funded by the TANG.NU-project funded by the Velux Foundation and by the Joint Doctoral Degree agreement between the Institute of Aquatic Resources (DTU Aqua) at Technical University of Denmark and the Norwegian University of Science and Technology (NTNU), Norway.
Statement of informed Consent, Human/Animal Rights: No conflicts, informed consent, human or
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2020, Advances in Botanical ResearchCitation Excerpt :In this new sporophyte seeding technique, the selection of a suitable twine was essential for the sucess of juveniles development (Kerrison et al., 2019). For Palmaria palmata cultivation, new hatchery strategies to improve seedling have being develop based on Germination, Maceration and Agitation (GMA) method - from release and handling of tetraspores to produce seedlings (propagules) on cultivation substrate (Schmedes, Nielsen, & Petersen, 2019). For the production of those seaweeds of mass cultivation by propagules, such as Gracilaria, for agar and, Kappaphycus and Eucheuma, for carrageenan extraction, several methods have been traditionally employed for rapid biomass production.
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