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Investigating hatchery and cultivation methods for improved cultivation of Palmaria palmata
Schmedes, Peter Søndergaard
Publication date: 2020
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Citation (APA): Schmedes, P. S. (2020). Investigating hatchery and cultivation methods for improved cultivation of Palmaria palmata. DTU Aqua.
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DTU Aqua National Institute of Aquatic Resources
Investigating hatchery and cultivation methods for improved cultivation of Palmaria palmata
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2 Contents
Preface 5 Acknowledgements 7 Referat (danish summary) 8 Summary 10
PART 1 1. General introduction 13 1.1 Brief introduction to seaweeds 13 1.2 Palmaria palmata (dulse) 15 1.3 The use of seaweeds 17 1.4 Farming systems 21 1.5 Cultivation and productivity of seaweeds 24 1.6 Previous trials of cultivating P. palmata in Europe – scoping the challenges 26
2. Aims and work flow of the thesis 27 3. Synopsis 29 3.1 Access to P. palmata tetraspores – by field collection and induction of fertility 29 3.2 Spore release and settlement - testing new hatchery methods to improve P. palmata cultivation 31 3.2.1 Early growth of seedlings 34 3.3 Grow out of P. palmata 36 3.3.1 Sea-based grow out from seeded substrates 36 3.3.2 Land-based grow out using vegetative propagation of biomass 37
4. General discussion 40 4.1 Is year-round spore induction reliable for future hatchery production of P. palmata?40 4.2 How to secure seedstock in future P. palmata cultivation? 42 4.3 What are the optimal methods for P. palmata spore release and seeding? 43 4.4 How to improve grow out of P. palmata?44 4.5 Land-based grow out 47 4.6 Selection of ecotypes and breeding 48 4.7 Overall conclusion 49 Appendix 1 50 5. References 52
PART 2
Paper 1 Reproductive phenology and investigation on the induction of fertility in 65 Palmaria palmata (Rhodophyta) Paper 2 Improved Palmaria palmata hatchery methods for tetraspore release, 89 even settlement and high seedling survival using strong water agitation and macerated propagules Paper 3 New hatchery methods for efficient spore use and seedling production 99 of Palmaria palmata (dulse) Paper 4 Productivity and growth rate in Palmaria palmata affected by salinity, irradiance and 111 nutrient availability – the use of nutrient pulses and interventional cultivation Paper 5 Seabased cultivation of Palmaria palmata - the effect of seeding method, substrate type, 135 deployment configuration, and farm location
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4 Preface
This thesis was submitted as part of the requirements for the degree of Doctor of Philosophy (PhD) at the Technical University of Denmark and the Norwegian University of Science and Technology (NTNU). The research presented in this thesis was carried out at the Danish Shellfish Centre (DSC, Nykøbing Mors), National Institute for Aquatic Resources (DTU Aqua) under the supervision of researcher Mette Møller Nielsen (DSC), professor Jens Kjerulf Petersen (DSC) and co-supervised by Kjell Inge Reitan at the Norwegian University of Science and Technology (NTNU). An external research stay was undertaken at NTNU, in association with the seaweed group at SINTEF OCEAN, Trondheim, Norway. Funding for this PhD project was provided by DTU Aqua and NTNU, the Velux Foundation and the Norwegian Research Council through the three projects “MacroSea”, “MIND-P”, and “Tang.NU”. The research stay was partly funded by economic support from the Augustinus Foundation
I have always lived by the coast and been fascinated plant growth. I come from a family of farmers and doctors, so the interest for growing crops and natural sciences is in my blood and at a young age, I enjoyed experimenting with farming vegetables and early on took the lead in my family garden. In 2007, I began my education to become a biologist at Aarhus University, which has given me a solid understanding of ecosystem services - and also a concern for natural resource depletion by human consumption. This coupled with my eagerness for hands-on projects and my ability to think outside the box, lead me to pursue a master’s project in sugar kelp cultivation. This was the beginning of a growing fascination for optimizing seaweed cultivation, boosted by the movement for sustainability and seaweeds as a new superfood being on the raise. My work at the aquaculture company Hjarnø Havbrug during 2013-2015 further drove my interest in cultivating seaweeds. Here I was employed together with Teis Boderskov, my good friend and companion during my master´s education, to start up the first organic large-scale farming of sugar kelp in Denmark. At the same time, my interest in cultivating another seaweed species, palmaria palmata, was growing. I read the book Seaweeds – vegetables of the sea by professor in gastro physics Ole G. Mouritsen and at a conference, I listened to one of talks of Ole G. Mouritsen, where he expressed his culinary fascination for the use of this red seaweed species in the new Nordic cuisine. I was eager to contribute further to the solution for a future “green deal” regarding food security by promoting a sustainable production of a low trophic marine species, and to take the challenge of farming this red algae species, known for its good taste, but also notorious for the difficulty of cultivating it. It was therefore a quite easy decision to leave my former job as a state-employed biologist, when I in 2016 was fortunate to get the possibility of conducting a PhD project with the aim of developing methods to grow Palmaria palmata.
The present PhD project focuses on improving the biological understanding and practical methods in the hatchery and grow out to enable an optimization for its future cultivation. Part 1 of the thesis contains a general background to introduce seaweeds and their aquaculture, as well as a synopsis of the main results of the PhD study and a discussion to elaborate on the status and future trajectory for the aquaculture of P. palmata.Part 2 contains five journal manuscripts of which two are published in international peer-reviewed journals, one is under review for publication, and two are in preparation for submission.
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Schmedes , Mette Møller Nielsen, Jens Kjerulf Petersen Technical University of Denmark, DTU Aqua, Øroddevej 80, 7900 Nykøbing Mors, Denmark ARTICLE INFO ABSTRACT Keywords: Cultivated Palmaria palmata is highly valued as a nutritious source of biomass. Yet, current hatchery techniques Palmaria palmata using tetraspores show low spore-to-seedling efficiency, normally imposing a high requirement of sori for Spore release seeding in large-scale cultivation, and pointing to a need for developing current hatchery techniques. This study Spore settlement shows new hatchery strategies to improve tetraspore release, efficiency of spore use as well as seedling dis- Seeding tribution on seeded substrates for P. palmata cultivation, based on germination, maceration and agitation (GMA- Seedling survival method). Re-attachment − We showed increased spore yield by using high-agitated sporulation tanks (67,906 ± 11,303 spores g FW 1) − compared to calm water (17,889 ± 3652 spores g FW 1). In addition, twine substrates cultured in high water − agitation (2.5 L air min 1) resulted in highest settlement and dispersal of spores and seedlings compared to non- agitated cultures. Using alginate coated twine showed no effect after 70 days nursery growth, despite higher initial spore density after a 22 days spore release phase in some treatments. Spore release time did not affect spore yield when comparing 1 and 3 h, whereas the yield increased during long-term sporulation (22 days) in some treatments. Released tetraspores settled in dense aggregates that ger- minated into a mixture of spores and seedlings (propagules) during 30 days and showed an ability of discoid re- attachment and growth after a maceration pretreatment. Here, the level of water agitation affected the re- attachment success and 39% of the added seedlings reattached after 14 days of nursery. The cultivation stra- tegies presented here provide a way to increase the overall spore-to-seedling survival and might serve as a new seeding strategy for P. palmata. Present findings are important knowledge in the quest of optimizing large-scale hatchery production of P. palmata. 1. Introduction on bottom-laid structures, such as mollusks valves or nets. Yet, the exploitation of seaweed in Europe is 99% based on harvest from wild Interest in cultivating of Palmaria palmata Linnaeus O. Kuntze (dulse stocks [5]. In Europe, Irish seaweed companies contributed with 99% seaweed) in European countries for human consumption and feed in- (< 100 t FW) of the annual harvest of P. palmata in 2011 [11]. P. pal- gredient, has increased as knowledge about its nutritious and potential mata is dried and retailed for high-valued niche products at prices health benefits is increasingly valued [1–5]. Globally, seaweed culti- reaching ~245 € per kg dry weight (DW). Besides a high content of vation is the fastest growing aquaculture sector and provides a low essential minerals and rich flavor [1,12,13] and relative high content of trophic and nutrient extractive source of biomass [5–7]. Several red crude protein of up to 19.3–35% [4,14], P. palmata has been char- algae species have been cultivated at small to larger scales in open acterized as promising diet supplement for humans and salmon feed water, such as Sarcothalia atropurpurea, Gracilaria lemaneiformis and due to the content of bioactive peptides, protein hydrolysates and an- Pyropia umbilicalis (former Porphyra umbilicalis) to secure a source of tioxidants [2,15,16]. As demands for P. palmata increase, focus on raw material for agar and carrageenan extraction as well as food, re- cultivation rather than wild harvest of this species is important to avoid spectively [8–10]. Those studies used spore inoculation methods and a diminishment of wild populations and to secure a sustainable crop period of calm water to promote settlement of tetra-, and carpospores production [1,17]. Cultivated P. palmata requires a relative high Abbreviations: psu, practical salinity unit; DW, dry weight; FW, fresh weight ⁎ Corresponding author. E-mail address: [email protected] (P.S. Schmedes). https://doi.org/10.1016/j.algal.2019.101494 Received 2 October 2018; Received in revised form 28 March 2019; Accepted 3 April 2019 $YDLODEOHRQOLQH$SULO (OVHYLHU%9$OOULJKWVUHVHUYHG 90 P.S. Schmedes, et al. $OJDO5HVHDUFK Fig. 1. Schematic illustration of two hatchery methods for P. palmaria. Top: Schematic illustration of normal hatchery steps cultivating P. palmata (A–D). A: Zoom-in on fertile tetrasporophytic frond (sori) containing tetrasporangia, each with four tetraspores. B: To inoculate substrates, a high amount of sori is normally used to cover the substrates during sporulation. C: Male gametophyte (seedlings) germination after few days while females remain microscopic or die off (red cross). D: After 1–3 months of nursery, male seedlings are visible on twine substrate. Bottom part: `Germinate-macerate-agitate´ (GMA) method (E–H). E: Released tetraspores settle in dense aggregates due to mucilage. F: Spore aggregates washed and transferred for germination in petri dish. G: To dislodge spores and tiny seedings after germination, aggregates were subjected to maceration to obtain a solution of propagules (spores and tiny seedlings). H: Adding a certain volume to the agitated inoculation tank provide dispersal of propagule settlement. Substrates (green) orientated in near-vertical position to increase space efficiency. Photo-credit: Werner & Dring, 2011 (B.), Constanza Chianala Cerda (C.). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) nitrogen availability during growth to maintain the dark-red color due substrate has been used in the seeding phase (B). However, this impose to phycobilliproteins [18], which is an important trademark for the a high demand of sori [26]. Thus, the ratio between the amount of sori species as commercial product. This have led to the idea of farming P. needed to seed substrates and the harvest yield is high, which indicate a palmata in nutrient riched areas [7,19,20]. However, in near-coastal low seeding efficiency. Using this method, Werner & Dring (2011) es- waters, like Danish inner waters where nutrient levels may be elevated timated a requirement of 135 kg of fertile plants to seed 70 (1.2 ∗ 3m) due to run-off and fish farms, water bodies show great spatial and to obtain an extrapolated harvest yield of 1.8 ton FW. After settlement, temporal variation in salinity [21], even down to 15 psu (National half of the tetraspores germinate into male gametophytes (seedlings), monitoring survey data, NOVANA). Despite the potential of cultivating whereas the other half (female gametophytes) remain microscopic or in nutrient-rich semi brackish waters [22], the growth of P. palmata die off (C) as no fertilization step to initiate the development of new seedlings in low saline waters, as inner Danish waters, is yet to be in- tetrasporophytes is implemented in the normal cultivation practice. vestigated. Young male seedlings are typically nursed for 1–5 months before de- Open-sea cultivation of P. palmata requires a land-based hatchery ployed at sea (D). After 5–8 months of growth at sea, biomass is ready phase to produce seeded substrate, like twine or textile, using released for harvest in late spring when sea temperature is still within the op- meiotic tetraspores as seeding material [17,23]. However, as of yet, timal range for growth (6–17 °C) [14,24]. existing seeding and hatchery methodologies for large-scale cultivation Agitation mediates a physical disturbance at the water-sporangia are suboptimal and need further development [24]. The normal interface, which likely enhance sporangia wall rupture [28] and aug- hatchery process can be divided in three important steps: spore release, ment the spore release rate. In addition, the risk of contamination in- spore attachment and seedling growth (Fig. 1A–D). creases as fertile tissue remains in the sporulation tank for several days Tetrasporophytes release tetraspores during fall and winter from [31]. Thus, agitation of water might be important for a faster spore mature fertile tissue (sori) [17,25] (A). Current seeding methodology release rate and seedling survival in batch cultures. Likewise, water for P. palmata is based on a gravity principle due to the relative large agitation might increase evenness of settled spores as it simulates the size (Ø~30 μm) and non-mobility of the tetraspores [26,27]. By this conditions with moderate exposure and strong currents found in the method, fertile sori submerged in inoculations tanks release tetraspores, lower intertidal and sublittoral habitat where P. palmata live. In this which settle during few days [17]. The spore release is highly un- habitat, P. palmata is often observed in high densities on stipes of La- predictable as ripeness and rupture of the tetrasporangia varies even minariales [25], suggesting that the stipe surface provides a higher within tissue sections [25,28,29]. Furthermore, the sticky nature of P. chance for spore settlement, survival and growth. After release, spores palmata tetraspores often result in settlement of nested spore aggregates settle and attach to kelp stipes mainly as result from higher encounter [29] of high density on the substrate or on bottom of the tank [24]. As a rate enforced by small-scale water eddies [32,33], though other factors, result, the spore density on substrates can be highly variable and cause such as stipes surface roughness and herbivory might also be important low quality seedling lines [24,30]. To compensate for the low spore use [34,35]. The stipes of kelps contain an extracellular alginate matrix to efficiency inherent in this method, a 1:1 areal coverage of sori-to- which sticky tetraspores might show preference of settlement attributed 91 P.S. Schmedes, et al. $OJDO5HVHDUFK to the outer layers of mucilage sheath and the adhesive mucilage ve- at 5 °C. Raft counting of all subsamples was done using a 100 x mag- sicles [28,36]. Mimicking an alginate surface on cultivation substrates nification stereomicroscope. might enhance the settlement and survival of P. palmata seedlings. Based on the previous findings and challenges as summarized 2.2. Preference of spore settlement and seedling survival (exp. 2) above, we aimed to investigate a modified strategy (Germinate-mace- rate-agitate; GMA) to optimize release and handling of P. palmata tet- The effect of alginate coated twine and three levels of agitation on raspores to produce seedlings on cultivation substrate, as shown in spore settlement and seedling survival was assessed during a 70 days Fig. 1(E–H). First step was to test the effect of agitation and spore re- experiment (8th of December 2016 to 17th of February 2017). lease duration on spore yield (E). Aggregates of released tetraspores Polypropylene 3-cordel twine (PP, Ø = 6 mm) was cut into 18 pieces of were transferred and germinated in petri dish (F) obtaining a mix of 10 cm and half were coated 24 h before the experiment with a ~1 mm spores and seedlings (propagules). After germination, the aggregates thick layer of alginate glue and tested against non-coated twine (con- were subjected to maceration as pretreatment to dislodge propagules trol). The glue was made by mixing 2 g of alginate powder (TYSA LN (G) and used as seeding inoculum in agitated tanks to test the ability of 80, Lot. no: 7152 NIM, EUROGUM A/S, Denmark) in 150 mL fresh propagules to re-attach on substrates. Three levels of agitation and two water. Nine gently cleaned fertile fronds were divided between nine types of twine were tested. After settling propagules were denoted plastic beakers with 400 ml seawater together with both types of twine ‘seedlings’ (H). Finally, we tested the effect of alginate-coated substrate tilted towards the surface at a 20° angle. To enhance spore liberation and the level of water agitation on tetraspore settlement density and and dispersal, the sori were fixed vertically in the beakers with constant salinity effect on seedling growth. agitation of water. Three levels of agitation were provided in triplicates − − − by aeration of 0 L min 1, 0.5 L min 1 and 2.5 L min 1. The spore re- 2. Material & methods lease phase ended after 22 days, where the sori were removed from the beakers and each pair of twine was transferred to clean beakers. From Sterile P. palmata (n = 50) were collected by divers the 23rd of day 11 to day 70, the number of settled spores (per g FW) was counted August 2016 near Fyns Hoved, Denmark (55.610895°N 10.594308°E) at weekly using a magnification microscope. From day 28, the density of 4–5 m depth and water temperature of 15 °C. The salinity of the upper seedlings was start counted. At high numbers of settled spores and water column at the collection site varies seasonally between 14 and seedlings, the average count on three subparts (1 cm) was used to es- 27 psu (National monitoring survey data, NOVANA) but was not mea- timate the density. The experimental conditions was 10 °C with a sured at sampling time. The plants were transported to indoor tank photoperiod of 10:14-h light:dark (35 EPAR) to simulate spring growth facilities and cultured in 400 L aerated filtered seawater (1 μm) at 10 °C season. Weekly water renewal was spiked to ~88 μM N by adding and exposed to 12:12-h light:dark regime with a surface irradiance of 0.4 ml of F/2+ growth medium in each beaker. −2 −1 80 PAR (EPAR; μmol photons m s ). Nutrients were added − (0.20 ml L 1 seawater) weekly to spike the nitrogen concentration to 2.3. The effect of twine type and agitation on propagule re-attachment (exp. ~175 μM N (N/P = 25) using growth media F/2+ (Varicon Aqua High- 3) cell F2P). After one month, the light:dark regime was changed to 8:16-h to simulate winter conditions and plants were kept under those con- To optimize the spore use, the ability of released propagule ag- ditions until start of experiments in October, January and February. gregates to re-attach and grow was tested in experiment 3 and 4 (see Four experiments were set up to investigate the mentioned challenges. 2.4). In exp. 3, propagule aggregates were macerated and used as All experiments were performed at 10 °C with down-welling irradiance seeding inoculum to test the effect of twine type and water agitation provided by pairs of fluorescent tubes (54 W, warm white, T5 Philips) (Fig. 1F–G). On February 10 2017, two pieces of fertile sori (2 ∗ 4 cm) and the distance to experimental tanks and neutral screens was used to were excised and kept in a 0.5 L container with gently aeration and adjust EPAR (measured by use of a planar LiCOR sensor). Filtered checked for released spores daily until February 17 where a spore ag- (0.2–1 μm) and sterilized (150W-UV) natural seawater of 26–28 psu gregates were visible on the bottom. The aggregates were divided into was used in all experiments. two petri dishes (25 ml) and left to germinate for 30 days at 5 °C, 7 ± 2 EPAR (10:14-h light:dark) with no nutrients added. Water was renewed 2.1. Spore release (exp. 1) twice during the period. After 30 days, the propagules were dislodged, poured into a glass beaker and macerated using a kitchen blender for A spore release experiment was conducted on the 10th of January one minute (Fig. 1G). The beaker was added 100 mL of clean seawater 2017 testing two levels of water agitations (calm water vs. agitated (total 150 mL) after which 15 mL of the propagule solution were added water) and durations (1 vs. 3 h) to assess the difference in spore yield to each of nine open plastic beakers with a total water volume of and spore release rate. Three plants (n = 3, 6–12 cm) were cleaned with 400 mL (0.2 μm filtered). The beakers contained a piece of either PP 1 μm filtered-sterilized seawater using paper towels before they were twine (10 cm, Ø = 6 mm) or polyvinyl Kuralon twine (10 cm, left to partially dehydrate for 20 h between layers of paper towel in a Ø = 2 mm). Three days before the experiment, all twines were washed dark room at 5 °C [26]. Subsequently, four sori pieces of each 1 cm2 and soaked in fresh water and left to dry before use. Each twine was (corresponding to approx. 0.05 g FW) and similar appearance were cut placed in a vertical position in the beaker with approximately 20° angle from each plant, and divided in 12 glass beakers containing 50 ml clean to the light source. Winding the soft Kuralon twine three times around a seawater to make up triplicates of the four treatments. Randomly cable tie secured its vertical position in the containers. Cultures were aligned beakers were exposed to irradiance of 25 EPAR at 10 °C, ac- kept at 5 °C, 10:14-h light:dark regime of 30 ± 5 EPAR. For the PP − − cording to [24,37]. Water agitation was mediated by air bubbles of twine, two levels of agitation (0.5 L min 1 and 2.5 L min 1) was ap- − 2.5 L air min 1 in half of the beakers whereas no aeration was applied plied in triplicates, whereas for the Kuralon twine agitation was only − in the other beakers. Agitation level was estimated prior to experiments applied at one level (2.5 L min 1). To evaluate the effect of treatment, by adjusting the airflow required to replace an equivalent volume of the density of spores and seedlings was counted at day 3 (settlement) water per minute in a water-filled glass beaker submerged upside down and at day 14 (attachment) using a stereomicroscope, as described in into a larger beaker. After 1 or 3 h respectively, the spore yield was exp. 2. estimated by counting three subsamples of 5 ml for each replicate. Before subsampling, tissues were gently removed and the water in the 2.4. Effect of salinity on growth of re-attached seedlings (exp. 4) beakers was stirred and aerated to obtain a well-mixed solution. Each subsample was pipetted into gridded well-plates and left to settle for 5 h P. palmata seedlings were cultivated at four different salinities (15, 92 P.S. Schmedes, et al. $OJDO5HVHDUFK 20, 25, and 30 psu) to assess the effect of salinity on early growth. The salinity range is realistic to simulate Danish inner surface waters in which seaweed cultivation takes place. The seeded twines were pro- duced based on re-attachment of germinated propagules, as described in exp. 3. On November 23, a one-day-old spore aggregate was trans- ferred to a petri dish and germinated until December 21st. After ma- ceration, the solution was poured into an open plastic beaker con- taining 400 mL seawater and five pieces of PP twine (10 cm, Ø = 6 mm) in a 20° tilted position. The water was agitated by aeration (2.5 L air − min 1) to promote dispersal of propagules before settlement. After 14 days of cultivation, re-attachment of solitary and nested spores and tiny seedlings on all twines was confirmed under the stereomicroscope. Seedlings were then pre-cultivated at 26 ± 1 psu for 41 days, before transferred to different salinity treatments. Then, each twine was cul- tivated in separate beakers containing the different salinities for 57 days with weekly water renewal. The different salinities were obtained by adjusting natural filtered (0.2 μm) and sterilized seawater by MilliQ water or Red Sea Coral salt powder the day before use and was added nutrients to spike concentration to ~88 μM-N. During 2 month of cul- ture, the seedling length was measured six times on the eight longest individuals from each beaker, according to [26]. Fig. 2. Spore yield of P. palmata as a function of spore release time (1 and 3 h) − − and level of water agitation (0 L min 1 and 2.5 L min 1). Data are presented as 2.5. Data analysis and statistics mean ± SE (n = 3). The Shapiro-Wilk W test for normality of residuals and Levene test represents two potential deployment dates and seedling density at de- for homogeneity of variances and outliers was applied for all datasets ployment day has great effect on the final biomass harvest [24]. using JMP 13 (SAS). The effects of spore release time and agitation on Within the first 22 days (spore release phase), spore settlement was spore release yield (exp. 1) were compared by a two factor ANOVA evident on the pairwise-cultured twines at all agitation levels (Fig. 3). (2fAN, α-level = 0.05) followed by a post hoc t-test (pooled error term). At day 22, the end of spore release phase, there was no significant The effects of twine coating and agitation on final density (day 70) of differences between non-coated and coated twines for any of the agi- tetraspores and seedlings (exp. 2) were analyzed by the Wilcoxon Test tation levels (ChiSq, P = 0.27). Data from both coating types were based on ranks followed by Steel-Dwass all pairs test due to non-in- therefore pooled in the following analysis. The spore release rate was dependence of the pairwise cultured twines. The dataset were log significantly higher in high-agitated cultures (P = 0.017) compared to (Y + 1) transformed to avoid zeroes, impose homoscedasticity but not semi-, and non-agitated cultures, evaluated by the amount of settled normality. When pooled, datasets were compared by use of the 2-tailed spores at day 22. Spores settled evenly in top and bottom of both types signed-rank test. In exp. 3, the main factors (twine type and agitation) of twine (data not shown). Comparable with the spore release phase, no were unbalanced, but data came from a homoscedastic population. The differences between coated and no-coated twine was found from day 22 mean density of re-attached propagules (spores and seedlings) on day 3 and onwards (P = 0.51). Data from both coating types were therefore and day 14 was compared using the Wilcoxon Test based on ranks pooled in the following analysis. At day 70, the high-agitated cultures followed by Steel-Dwass All Pairs comparison test. In exp. 4, the eight maintained a significantly higher spore density compared to the lower longest seedlings on each twine were regarded as treatment replicates levels of agitation (P = 0.004), whereas the semi-, and non-agitated as no density-depended effect was assumed by use of tiny seedling sizes, cultures were not significantly different. The density of settled spores in even light availability, weekly water renewal and high nutrient con- high-, semi-, and non-agitated cultures being 272 ± 75, 38 ± 26 and centration [26]. The effect of salinity on mean seedling length at the 2 ± 1 spores/twine, respectively. final date was assessed using one-way ANOVA followed by Tukey- In general, the seedling density increased in high-agitated cultures Kramer HSD test on log-transformed data converted to micrometers in contrast to lower agitated cultures (Fig. 3). Twine coating showed no (log(Y*1000)). When variables did not show significance, the data was significant effect on seedling densities after 70 days (P = 0.27). Thus, pooled for further comparison (Student's t-test). All data are presented data from both coating types were pooled in the following analysis. as mean ± SE if not stated otherwise. Water agitation significantly affected the final seedling density (ChiSq, P = 0.0024; day 70), where the high-agitated cultures showed seedling 3. Results densities of 141 ± 37 seedlings/twine compared to 11 ± 6 seedlings/ twine in the semi-agitated cultures (P = 0.0224) and 3 ± 2 seedlings/ 3.1. Spore release (exp. 1) twine in the non-agitated cultures (P = 0.0121) (Fig. 4). During the phase of spore release, water agitation mediated a sig- nificant effect (P = 0.0308) on the spore yield, whereas the effect of 3.3. Effect of twine type and agitation on propagule re-attachment (exp. 3) 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 re- The seeding material obtained by macerating the propagule ag- − sulted in a spore yield of 67,906 ± 11,303 spores g FW 1 compared to gregate following 30 days germination (propagule solution) contained − 17,889 ± 3.652 spores g FW 1 in calm water for 1 h. No interaction both single and small clumps of spores and tiny seedlings (2–5 in- between agitation level and spore release time was found (Fig. 2). dividuals) with a concentration of 37 spores/mL and 15 seedlings/mL of both solitary and nested propagules. The mean initial length of 3.2. Preference of spore settlement and seedling survival (exp. 2) seedlings was 0.6 ± 0.2 mm (n = 10). The settlement density of spores at day 3 (Fig. 5a) was similar be- To evaluate treatment effects, we compared only the final counts of tween all treatment groups, despite of different agitation levels the spore release phase (day 22) and after nursery (day 70), as this (28 ± 5 spores, pooled data). At day 14, the spore density was 93 P.S. Schmedes, et al. $OJDO5HVHDUFK Fig. 3. Temporal development in spore densities on alginate coated and non-coated twine as effect of agitation. Pairs of the different twines were seeded in the same inoculation tanks. Sori were removed at day 22. Data points represent the mean count of spores ± SE (n = 3). significantly lower on the Kuralon twine than on PP twine with same groups (ChiSq, P = 0.0054), the low agitated group being significant level of agitation (P = 0.016, students t-test) whereas the two agitation lower than the two high-agitated groups, and the high-agitated groups levels applied for the PP twine did not impose any differences in spore being independent of twine type. High agitation promoted a settlement density. density of 58 ± 12 seedlings (pooled data, n = 6) compared to 10 ± 4 Seedling density at day 3 differed significantly between the three seedlings in the low-agitated PP group at day 3. Similar to day 3, the Fig. 4. Temporal development in seedling densities on alginate coated and non-coated twine as effect of agitation. Pairs of the different twines were seeded in the same inoculation tanks. Sori were removed at day 22. Data points represent the mean count of seedlings ± SE (n = 3). 94 P.S. Schmedes, et al. $OJDO5HVHDUFK Fig. 5. Density of settled (day 3) and attached (day 14) P. palmata spores (A) and seedlings (B) obtained from inoculating two types of twine; Kuralon (Kur) and polypropylene (PP) in seeding tanks with a solution of germinated and macerated propagules. PP twine was cultured at two levels of water agitation by aeration of − − − 0.5 L min 1 or 2.5 L min 1, while Kur was cultured at 2.5 L min 1 only. Data represents mean ± SE (n = 3). density of seedlings were significantly higher in both high-agitated seedlings (n = 8) was significant (P = 0.0039) longer at 15 psu groups compared to the low-agitated (P = 0.0053) at day 14, and high- (2.0 ± 0.3 mm) and 20 psu (1.5 ± 0.4 mm) compared to the 30 psu agitated groups not being significantly different. The high-agitated culture (0.67 ± 0.54 mm), however not significantly differing from groups showed a density of 53 ± 5 seedlings (pooled data, n = 6) each other (Fig. 6). At 15 and 20 psu, the specific growth rate was 2.4% compared to 20 ± 3 seedlings for the low-agitated group at day 14. per day during ~2 months, using the equation (Ln(L2)-Ln(L1)/t2-t1), Combining counts of propagules in the inoculum with spore and which is comparable to previous reports [14,26]. The mean seedling seedling counts (Fig. 5), the success rate of reattachment (and survival) length of the 25 psu culture was significant smaller (P = 0.049) com- of settled spores and seedlings were estimated to 5% ((28 sporesATTA- pared to 15 psu, but not the 20 psu culture (P = 0.59). CHED / 37 spores/mLINOCULATION ∗ 15 mL) ∗ 100%) and 39% ((58 seedlingsATTACHED / 10 seedling/mLINOCULATION ∗ 15 mL) ∗ 100%), re- 4. Discussion spectively. The main objective of this study was to investigate how modified 3.4. Re-attached propagules cultured in a salinity gradient (exp. 4) conditions in each hatchery step, e.g. tetraspore release, settlement, and early stage growth can improve seedling production of P. palmata.Ina The average seedling length increased significantly (P < 0.005) stepwise approach, we have demonstrated a seeding method for P. during the experiment in all salinity treatments (Fig. 6), the overall palmata based on water agitation and maceration of germinated pro- pattern being a continuous growth until the end of the experiment at pagules for dispersal and re-attachment to cultivation substrate. The salinities of 15 and 20 psu, whereas growth declined slightly at the end novel findings indicate a potent discoid adhesion capability of the for cultures at 25 and 30 psu. young spores and seedlings after dislodgement which was enhanced by After 57 days of cultivation, the maximum average length of maintaining high water movement during inoculation. In comparison to protocols for cultivating other thalloid red macroalgae, such as Gracilaria chilensis [38] and Sarcothalia crispata [39], calm conditions are often described to allow tetra-, and carpospores to settle on hor- izontal surfaces. In addition, this method potentially shows a way for spore aggregates to be stored before maceration using low light and low temperature conditions (7 ± 2 PAR, 5 °C), which could be a mean of reduced risk of epiphyte fouling [14]. Based on collection of fertile sporophytes or by the suggested year-round manipulative induction of sori tissue [40] this method might be used to extend the seedling pro- duction season by control of the germination rate of tetraspores into seedlings. Using macerated propagules, the survival of settled spores and seedlings was estimated to 5% and 39% respectively, after 14 days of nursery cultivation. The lower survival of spores may however be an effect of spores germinating into seedlings during the 2 week nursery phase, which falsely affects the survival rate of spores negatively. Thus, as an alternative to the normal seeding method (Fig. 1A–D), which promotes a patchy spore density and a considerably spore loss during sporulation, the present maceration-method (Fig. 1E–H) suggests a way to increase spore utilization and a way to control seeding density. 4.1. Spore release Fig. 6. Growth in seedling length (mm) measured weekly or biweekly on the eight longest seedlings, obtained by the re-attachment method, and cultured The spore release yield did not differ on a short-term basis (1 vs. 3 h, during 57 days on PP twine (Ø = 6 mm) at different salinities. Data points re- exp. 1), whereas a generally increasing spore density during the 22-day presents mean ± SE (n = 8). spore release phase in exp. 2 indicates an effect of long-term spore 95 P.S. Schmedes, et al. $OJDO5HVHDUFK release. This suggest a different strategy for hatchery spore release in P. 4.4. The effect of salinity on early seedling growth palmata than for instance for brown macroalgae such as S. latissima, where a sufficient spores release is obtained within a short time-frame P. palmata seedlings cultivated at constant low salinity (15 psu) for [41] and pinpoints an important consideration for P. palmata cultiva- 57 days showed similar and even higher growth in length than higher tion. salinities (up to 30 psu). Similar growth response has been found in − The high spore release yield of 67,906 ± 11,303 spores g FW 1 other coastal macroalgae species; Ulva intestinalis (former Enteromorpha − − (corresponding to 22,635 spores g FW 1 h 1) found in this study at intestinalis) and Fucus serratus both showing the highest growth rate high agitation (exp. 1) suggests that high-agitated water enhance between 15 and 20 psu [45,46]. This indicates an opportunity to cul- sporangial rupture and thereby increase spore release. Also, the release tivate P. palmata in areas of Danish inner waters where a salinity of rate found here was markedly higher than the estimated release rate of these levels often prevails. − − ~4 spores g FW 1 h 1 previously found by use of the gravity-based seeding method [24] where ~43.000 tetraspore released and settled 5. Conclusions during three days of sporulation. Several factors affect the spore release of fertile P. palmata, such as To establish commercial P. palmata cultivation, a viable hatchery season, geographical position and typography at collection site, which seedling production is crucial as tetraspores are short in supply, spore all affect the ripeness of sori [24,30]. This might explain the high usage has currently been suboptimal and seedling density affects har- variation in hatchery-based spore release yield of P. palmata in earlier vest yield. Based on this study, we suggest a new versatile strategy to − studies of 5.000–60.000 spores g FW 1 ( ± 30.000) [20,23]. optimize the use of P. palmata tetraspores based on germinated spore The positive effect of using relative strong water agitation during aggregates subjected to maceration to produce a solution of adhesive hatchery spore release, seeding and nursery phase might relate to the propagules showing the ability of discoid re-attachment on substrate. hydrodynamic forces found in the lower intertidal and sublittoral Besides, level of water agitation strongly affected the re-attachment of coastal waters which P. palmata inhabit. Similarly, the use of agitation macerated propagules and led to increased spore release and spore enhanced the release of haploid spores in other macroalgae inhabiting distribution on twine. Future large-scale P. palmata hatcheries may coastal waters, such as Alaria esculenta, Ulva lactuca and Porphyra um- benefit from this study, as a way to enhance spore-to-seedling efficiency bilicalus [10,42]. In these studies, water stirring also promoted spore-to- by use of Germination, Maceration and Agitation (GMA) for enhanced seedling germination. efficiency in seeding production. The methods allows for extended season of seedling production by slowly germinating collected spore 4.2. Spore settlement and survival aggregates. To take P. palmata cultivation to the next level, more re- search in optimal use of the multiple and discrete sporulation events of In the next step of the hatchery phase, water agitation significantly P. palmata including control the slow germination and storing, is enhanced spore settlement and final seedling density on twine when however still needed. Furthermore, to improve overall seedling density sori were left to inoculate for 22 days in the seeding tanks (Figs. 3–4), on cultivation substrates, future studies may investigate the optimal documenting the positive effect on seedling survival by water agitation. timing of a fertilization step to generate new sporophytes out of the Also, non-agitated cultures resulted in poor spore and seedling dispersal female spores. Salinity responses of growing seedlings might also be on twines (exp. 2) when using sori in seeding. However, the application important for future studies investigating site selection for cultivated P. − of even low level of water agitation (0.5 L min 1) was sufficient to palmata in brackish waters. distribute germinated spores for re-attachment, whereas the re-attach- ment and survival of seedlings increased with higher level of water Declaration of interest agitation (exp. 3) and reached a high survival rate (39%). This is in the high range of what has been found in other studies showing survival None. rates of 10% to 35% over the course of 7 days and 2 months during nursery phase, respectively [24,26]. Effectively, we suggest the use of Acknowledgements high-level agitation for spore release and propagule dispersal to lower the amount of collected fertile sori required for hatchery seedling We thank local scuba divers for collecting the P. palmata biomass production. and Kasper Lenda Andersen for tank maintenance in the hatchery. The study was funded by the TANG.NU-project funded by the Velux 4.3. Substrate coating Foundation and by the Joint Doctoral Degree agreement between the Institute of Aquatic Resources (DTU Aqua) at Technical University of Several cultivation trials have documented the hatchery steps for Denmark and the Norwegian University of Science and Technology cultivating P. palmata using the gravity-based seeding method (NTNU), Norway. [17,19,23,24,43], however, little improvement of spore-to-seedling Statement of informed Consent, Human/Animal Rights: No con- viability has been shown. The use of coating on cultivation substrate is flicts, informed consent, human or animal rights applicable. an emerging technology in hatchery seeding to lower the cost of hatchery operation [41]. For instance, mixing S. latissima gametophytes References and tiny sporophytes into a binder solution containing soluble algal sugars and applying it on cultivation substrates has shown to prevent [1] O.G. Mouritsen, C. Dawczynski, L. Duelund, G. Jahreis, W. Vetter, M. Schröder, On propagules to be washed off when deployed [42]. P. palmata is a the human consumption of the red seaweed dulse (Palmaria palmata (L.) Weber & Mohr), J. Appl. 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Mortensen, Remediation of nutrient-rich, brackish fjord water through 97 blank page 98 Paper 3: New seeding methods for efficient hatchery production 99 Journal of Applied Phycology https://doi.org/10.1007/s10811-019-01998-0 23RD INTERNATIONAL SEAWEED SYMPOSIUM, JEJU New hatchery methods for efficient spore use and seedling production of Palmaria palmata (dulse) Peter Søndergaard Schmedes1 & Mette Møller Nielsen1 Received: 20 August 2019 /Revised and accepted: 11 November 2019 # Springer Nature B.V. 2019 Abstract Palmaria palmata (dulse) is a high valued rhodophyte; nevertheless, its hatchery methods are underdeveloped. New hatchery methods are required to improve the spore use efficiency and seeding quality, which are important benchmarks for a viable cultivation. This study investigated a method using vertical seeding tanks (exp. 1), hemispherical agitation and flow-through conditions to improve spore dispersal on net substrates. Tanks were inoculated with different amounts of sori tissue, which sporulated in three consecutive seeding periods. The results demonstrated significant effect of seeding period where 5–15 g FW sori could be used to seed three nets (~ 126 m rope) over the course of 9 days providing a density up to 10 seedlings cm−1 after 32 days. The effluent spores were collected in detaining tanks and germinated into a propagule mix of female and male gametophytes. The propagule mix was efficient as a secondary seeding inoculum, as propagules were able to reattach to substrates up to 39 days after their release as spores (exp. 2). Adding male gametes to the propagule mix and spore seeded ropes was tested as a relevant hatchery step to activate female gametophytes and significantly resulted in more than a doubling of seedlings (exp. 3). This study present new methods and strategies to improve spore use efficiency and to obtain an equal spore dispersal on net substrates for hatchery production of P. palmata. Keywords Rhodophyta . Dulse cultivation . Spore dispersal . Fertilization . Hatchery . Tetraspores . Reattachment . Spore seeding Introduction received increasing attention in Europe (Edwards and Dring 2011;WernerandDring2011; Sanderson et al. 2012). The rhodophyte Palmaria palmata (L.) F. Weber and D. Mohr However, despite studies founding the essential understanding has traditionally been used for human consumption with records of the life cycle (van der Meer and Chen 1979; van der Meer and dating back to the ninth century (Mouritsen et al. 2013). The Todd 1980) and cultivating P. palmata from spores (Browne emerging evidence of its umami flavor (Mouritsen et al. 2013), 2001; Sanderson 2006;WernerandDring2011; Grandorf Bak bioactive and health properties of protein hydrolysates (Harnedy 2019;Schmedesetal.2019), the methodology for large-scale et al. 2014; Admassu et al. 2018), water extracts (Lee et al. hatchery production in Europe is still in its infancy. 2017), and biorefined compounds (Schiener et al. 2017)has Essentially, the use of spores for cultivation of P. palmata is renewed interest in the use of P. palmata, resulting in increased based on three steps: (1) the collection or induction of sori, (2) focus on its cultivation. P. palmata canbecultivatedintanks the release and dispersal of spores, and (3) the spore attachment using vegetative growth (Morgan and Simpson 1981;Pangand and growth, and it is the efficiency and success of these three Lüning 2004; Matos et al. 2006; Corey et al. 2014), but the steps that determines the usefulness of a given hatchery proto- prospects of cultivating the species from tetraspores have lately col. As the steps are interlinked, the initial handling of sori, which represent only 8–10% of the total frond area during the peak season of fertility (Werner and Dring 2011), is crucially * Peter Søndergaard Schmedes important. Currently, the most commonly used hatchery proto- [email protected] col to produce P. palmata seeded substrates is a flat tank using a 1:1 areal coverage of substrate with sori and a 3-day spore 1 Technical University of Denmark, DTU Aqua, Øroddevej 80, release duration. However, this method requires a large amount 7900 Nykobing Mors, Denmark of sori and, furthermore, results in poor dispersal and high 100 JApplPhycol mortality (60–90%) of the spores (Werner and Dring 2011). improvements. Experiment 2 covered step four, optimizing Also, by the using this method, it is often observed that a con- the use of detained spores, and finally, experiment 3 exploited siderable amount of the spores settle on tank surfaces instead of the opportunity to include fertilization as a means to improve the intended cultivation substrate (personal observation). spore use, addressing step five of hatchery improvement Taking into account these results and observations, it is techniques. clear that there is a bottleneck in spore use efficiency within P. palmata hatcheries. This particular matter was addressed in Experiment 1: spore seeding and sori use a recent study suggesting a GMA-seeding method (germina- tion, maceration, and agitation methods) as an alternative Nine conical tanks (polyethylene, 30 L) were set up as seeding seeding method for P. palmata (Schmedes et al. 2019). This tanks in a parallel flow-through system with a water flow of study found that germinated propagules (i.e., a mix of spores 0.5 L min−1 (Fig. 1a) in a 10 °C cold room. Air bubbles from and seedlings) of P. palmata were able to establish discoid the bottom (1.2 L min−1), bubbled through the sori package reattachment to a substrate after forced de-attachment and near the surface (Fig. 1c), generated a hemispherical circula- maceration, resulting in high settlement efficiency as well as tion of the water allowing the spores to disperse and aerated a high dispersal of the propagules. However, the extensive use the tank volume. Water for the entire system was circulated of this seeding method is still unknown. Besides the use of this from a reservoir tank with a circulation pump and exchanged seeding strategy, the use of male gametes of P. palmata to (10% of total volume) on a daily basis. No nutrients were fertilize female gametophytes has been suggested as a poten- added. From each seeding tank, an outlet, placed diagonal to tial hatchery step to optimize the hatchery production (Mine the inlet, led the effluent water directly into a 2-L spore- and Tatewaki 1994;LeGalletal.2004; Schmedes et al. 2019). detaining tank (SDT) (Fig. 1b). Here, effluent spores aggre- When using sori for spore-seeding, a mixture of male and gated on the bottom, while water was led out in the top and female spores develops after attachment; however, only the back to the reservoir tank through a nylon filter (15 μm) for males will develop into a harvestable thallus unless a fertili- UV treatment (60 W). Spores detained in the SDTs were used zation step, enabling zygote formation in the female gameto- in experiment 2. phyte, is included. Theoretically, this would double the seed- Twenty-seven pieces of net (polypropylene, 0.25 × 1.40 m, ling density, as the crustose-like female gametophyte will de- ø = 5-7 mm) were prepared as cultivation substrate by soaking velop into a sporophytic thallus after the zygote. Yet, this is them in lukewarm tap water a month before, then kept in clean still uninvestigated under relevant hatchery conditions. seawater for 7 days before use. At the experimental start, the Because of the continued challenges in producing nets were submerged in the seeding tanks as a two-layered net P. palmata sporophytes and gametophytes, this present study spiral (Fig. 1b). investigated hatchery methods and strategies based on Fertile Palmaria palmata tetrasporophytes and male game- Schmedes et al. (2019) to further improve the utilization of tophytes were collected January 4, 2019 in the intertidal zone spores for cultivation of P. palmata on substrates. Five strate- near Fornæs light house, Denmark (56.443534 N, 10.958985 gies to improve the hatchery protocol were tested: (1) a verti- E) and kept in running seawater (4 °C) in dim natural light cal seeding tank in flow-through condition agitated with air (~15 μmol photons m−2 s−1, PAR). On 10 January, 218 fertile bubbles to promote dispersal of spores; (2) the effect of dif- fronds were rinsed in 0.2 μm filtered and sterilized seawater ferent amount of sori during the seeding phase; (3) the effect (Fig. 2a) before being desiccated for 20 h at 5 °C in darkness. of using the same sori material in three consecutive seeding The presence of ripe sporangia was verified by inspection periods on the spore germination and seedling density; (4) the (Fig. 2b). Triplicates of three groups of sori amount (5, 10, use of detained spores as a propagule seeding inoculum, based or 15 g FW; fresh weight) were prepared as sori packages by on the GMA method; (5) The effect of fertilization on the placing the tissue between two layers of plastic net (15 × 15 number of attached seedlings under relevant hatchery cm; Fig. 2c). conditions. The experiment started January 11, 2019 by adding a net and a sori package (3 replicates of 3 sori amounts) to each of the nine tanks. The seeding ran over a course of 9 days, keep- Materials and methods ing the same sori package in the tanks for all 9 days but exchanging the nets with new ones every third day to test Three experiments were set up to test the five improvements in the potential of consecutive seeding periods (days 0–3, days hatchery techniques described above. Experiment 1 investi- 3–6, days 6–9) using the same sori. During seeding, the sur- gated a new flow-through seeding system for improved face irradiance was 15–22 μmol photons m−2 s−1, 10:14 h seeding dispersal, additionally using different sori amounts L:D. At the end of each consecutive seeding period, the nine in consecutive seeding periods as a means to improve sori nets were labeled and transferred to nursery tanks containing use. This experiment addressed the first three steps of hatchery 300 L enriched seawater (0.2 μm filtered), 10% strength of 101 JApplPhycol Fig. 1 a Parallel flow-through setup of conical seeding tanks a (30 L) used for experiment 1. b The sori packages were fixed centrally above the net spirals and 2 cm below water surface. Effluent spores were detained in spore-detaining tanks (SDTs). c Aeration (1.2 L min−1)fromthe bottom provided hemispherical water circulation and dispersal of released spores b c Fig. 2 a Fertile tetrasporophytic fronds were cleaned and the b presence of dark-red sporangia were validated (b). c Sori pack- ages (5, 10, or 15 g FW) were prepared by placing the sori be- tween two layers of green plastic mesh (n =3) a c 102 JApplPhycol Varicon Aqua Cell-Hi F2P, a F/2 nutrient medium with vita- blender. (2) A 10-mL subsample was transferred to a mins based on Guillard and Ryther (1962). Air stones provid- gridded Petri dishes (Fig. 3c) and added 20 mL ed water agitation in the tanks after 3 days. Irradiance was enriched (10% F/2) seawater. (3) The spores were kept 35 μmol photons m−2 s−1 (10:14 h L:D) but raised to at 5 °C and 5 ± 2 μmol photons m−2 s−1 (10:14hL:D) 50 μmol photons m−2 s−1 from week 2. for 3 days before five random fields (each 0.0035 mm2) were photographed and counted. The total number of spores was estimated by factor multiplying the mean Data acquisition −1 count of subsamples (STDVOLUME 10 mL subsample −2 The net-seeding efficiency using sori in the flow-through sys- and Petri dish area 0.0035 mm ). tem was calculated by Eq. 1: #spores on net Experiment 2: mixed propagules as seeding inoculum Net seeding efficiency ¼ 100 ð1Þ #Total sporesðÞ net þ detained In experiment 2, the propagules germinated from detained This was carried out by counting the number of attached spores from experiment 1 were tested as seeding inoculum, spores and seedlings on a 4-cm net subsample for each of the 9 according to the GMA method (Schmedes et al. 2019). After nets (n = 3) 3 days after each seeding period. Additionally, the subsampling for Petri dish cultures in experiment 1, the rest of nets were counted at days 19 and 32 after each of the consec- the macerated spore solution from the last seeding period utive seeding periods to compare spore and seedling density (days 6–9) was poured into a plexiglas containers (5 L), settled within and between groups over time. The subsamples were on the bottom (Fig. 4a) and cultivated at 20 μmol photons taken from the same position regarding the inlet and outlet of m−2 s−1 (adjusted to 5 ± 2 μmol photons m−2 s−1, after 10 days) the tanks on each net spiral. To verify an even spore dispersal and 5 °C. After 1 day, enriched seawater and germanium- on the entire net, additional subsamples were taken from the oxide (10% F/2 + 1 mg L−1 GeO) was added and after 6 days, 10 g sori batch in the first seeding period (days 0–3). From an air stone was put for aeration. The detained spores germi- each net (n = 3), three subsamples were taken from the bottom nated into a mixture of propagules (i.e., spores and gameto- and top part of the net (n = 9) and tested for unequal variance phytes) at these conditions before use. using Levene’s test. Similarly, by counting spores on subsam- Twenty-seven days after the initiation of days 6–9seeding ples (n = 21) from all around one net spiral in the same vertical period, some of the propagules on the bottom were dislodged level, the spore dispersal was assessed. By verification, we (Fig. 4b), suspended in 400 mL enriched (10% F/2) seawater, extrapolated the spore and seedling densities for each counting and macerated to break propagule aggregates (Fig. 4c) accord- day and calculated the germination success by Eq. 2: ingtoSchmedesetal.(2019). The propagules were photographed before and after the maceration treatment (Fig. #Seedlings GerminationðÞ¼ % 100 ð2Þ #Total propagulesðÞ spores þ seedlings 4d, e). A total of 20 mL of the propagule mix was added to each of 10 beakers (n = 10) containing 400 mL enriched (10% F/2) The total number of detained spores in each SDT seawater and a piece of rope (10 cm) standing upright in the after a seeding event (Fig. 3a) was estimated based on beaker to test the ability of the propagules to reattach. The rope subsample counting, by the following process: (1) A was made by untangling several net meshes and cut and cut into homogenous spore mix (Fig. 3b) was prepared by re- pieces. Immediately, 1 mL subsamples were withdrawn and the ducing the SDT volume, dislodging the spore aggregates concentration of macerated propagules was estimated to 259 ± and macerating the solution for 30 s with a kitchen 8sporesmL−1 and422±13seedlingsmL−1 (mean ± SE, n = a bc Fig. 3 a Dark-red aggregates of spores on the bottom of spore-detaining The number spores were counted in five random fields (each of tanks (STDs) accumulated during sporulation. b A well-mixed spore so- 0.0035 mm2) and used as a basis to estimate the total amount of detained lution was obtained by dislodging the aggregates and macerating them. c spores 103 JApplPhycol a b c d ef Fig. 4 a Detained and macerated spores of P. palmata at the bottom of a maceration. The solution was inspected before (d) and after (e) the mac- 5-L Plexiglas tank. b Plexiglas tank after dislodgement of propagules on eration treatment. f Single individual seedling displaying hair-like prolif- the left side of the tank. c Propagule mix after dislodgement and erations from the basal disc area. Scale bar represent 500 μm 4). The beakers were constantly agitated from the bottom via air Experiment 3: including fertilization in hatchery bubbles (2.5 L min−1) to promote the dispersal of the propagules. The effect of fertilizing female gametophytes was tested by After 3 days inoculation (“agitationperioddays0–2”), the adding male gametes to propagules cultured in petri dishes maximum density of attached spores and seedlings was esti- and spore-seeded ropes cultured in beakers. A solution of prop- mated by counting the three densest subparts (0.5 cm) for each agules (349 ± 26 propagules mL−1, n = 9, technical replicates) rope, after which the ropes were transferred to 20 L tanks at was prepared from the same detained spore batch as used in the similar conditions until they were counted again at day 10. experiment 2 by dislodging and macerating after 11 days of After transferring the ropes pieces out of the beakers, the germination. A total of 15 mL propagule solution was then propagule mix that maintained in the beakers under same con- poured into each of 18 petri dishes and filled with additional ditions until day 10, where concentration (mean of technical 10 mL enriched (10% F/2) seawater and kept in 15 ± replicate ± SE) of spores (237 ± 7) and seedlings (394 ± 9) was 3 μmol photons m−2 s−1 PAR (12:12 h L:D) irradiance at 5 °C. similar to the starting concentration (two-sampled t tests: t(4), At day 1, a solution of male gametes was prepared and p =0.086,p = 0.131, respectively). Then, new pieces of rope 15 mL of this solution was added to nine petri dishes, contain- were added for 3 days inoculation to see how long after the ing the propagule solution. Nine other dishes received 15 mL dislodgement and maceration the propagules would be able to seawater (10% F/2) as a control. The male gamete solution reattach (“agitation period days 10–12”). Also, at day 10, new was prepared by desiccating (3 h, dark at 5 °C) thirty fertile beakers (n = 10) were set up under same conditions using male gametophytic fronds (22.4 g FW; see Fig. 5a, b)before freshly dislodged and macerated propagules to see if the age being rehydrated (1 h, 5 μmol photons m−2 s−1 PAR) in an of the propagules (27 vs. 37 days) at dislodgement affected the agitated volume of 400 mL seawater + 0.4 mL GeO (1 g L−1). ability to reattach. The concentration of propagules (250 ± 20 The male gametes (Ø ~5 μm) were visible using high magni- spores and 396 ± 22 seedlings) was similar (two-sampled t fication (Fig. 5c, d). The concentration of gametes was esti- tests: t(4), p = 0.584, p = 0.341) with the other start mated to be ~ 1000 gametes mL−1, using a Neubauer cell concentrations. counting chamber. After adding, the cultures were mixed on 104 JApplPhycol a b c d Fig. 5 a Fertile male gametophytes inspected by stereomicroscopy. b Cross-section microscopy was used to verify reproductive appearance of the males. c, d Spherical male gamete released in a solution a stirring table for 30 min at 160 rpm. At day 4 and 6, the petri transformed to ensure homogeneity of variance and normal dish volumes were exchanged with new additions of male distributed residuals and ANOVAs were conducted to com- gamete solution (5 mL, ~ 106 gametes mL−1). pare the main effects. In case of none normal distribution or For each petri dish, spores and seedlings were counted in homogeneity of variance, the Wilcoxon two-sample test was five random fields (each 0.0035 mm2)atdays0,3,10and19 used. All data are given as mean ± standard error (SE), unless and the number of seedlings was adjusted to the spore count of stated otherwise. previous counting day. In addition, we assessed the effect of fertilization on the Experiment 1 In a factorial design, nets were manipulated to seeded rope pieces (exp. 2). This was carried out in beakers be in one of nine groups forming the combination of consec- (1.8 L) kept at 5 °C, in which 4 cm pieces of seeded rope were utive seeding periods (3 levels; days 0–3, days 3–6, days 6–9) tumbled by air bubble agitation (n = 11). The pieces were ex- and sori amount (3 levels; 5, 10, 15 g FW), and 2-way cised from “days 3–6and10gsori” nets (exp. 1) and nursed ANOVA, including the interaction term, using log- for 16 days at 10 °C, before being transferred to the beakers. transformed data was conducted to compare effects on spore This was followed by adding 40 mL solution containing male and seedling numbers attached to the nets, the number of gametes to the beakers, while a control group was added the detained spores in STDs, the spore-seeding efficiency, as well same enriched seawater (10% F/2) with no gametes (n = 11). as the germination success (%) between all groups, followed The number of spores and seedlings was counted at days 1, 3, by Tukey’s HSD post hoc test. The Wilcoxon two-sample test 6, and 12, and the seedling number was presented as relative was used to assess for any significant change in mean densi- to spore number. ties of spores and seedlings across the counting days within each factorial group. Statistics Experiment 2 Datasets were log-transformed to ensure normal For all data sets, Shapiro-Wilk test was used to check normal- distribution, but spore counts did not obey homogeneity of ity and Levene test was used to check variance homogeneity. variance (Levene, p < 0.0208). Hence, the Wilcoxon test was The analysis was carried out using SAS, JMP 13, using a used to assess for significant difference in mean settlement significance level of 0.05. When sufficient, data were log- density at each count day. 105 JApplPhycol Experiment 3 For the petri dish cultures, the effect of adding germination success (Fig. 6g–i)wassimilarforallgroups male gametes on the number of seedlings adjusted to the pre- (2-way ANOVA: p > 0.5606). On counting day 32, the vious spore count was compared using a two-tailed Student’s t spore settlement was still similar across all groups (2-way test (n = 11). For the rope cultures, the univariate repeated ANOVA: p > 0.1179) (Fig. 6a–c), whereas the number of measures ANOVAwas used to test effect of the between factor seedlings (Fig. 6d–f) was significant affected by seeding (two levels; male gametes vs. control) and the within factors period (2-way ANOVA: F2,8 = 6.1718, p = 0.0091), show- (time; four levels) on the adjusted number of seedlings. ing similar seedling density in second and third period Datasets were normal distributed and displayed equal variance (Tukey’s, p = 0.8110), both significantly higher than the after log-transformation. The Mauchly’s sphericity test: χ2 first seeding period (Tukey’s, p = 0.0105, p =0.0383,re- (5) = 8.3513, p = 0.1379 allowed to report the p value of the spectively). At day 32, the average seedling density was F test. 36 ± 8 seedlings per 4 cm. However, the germination suc- cess (Fig. 6g–i)upto50–80% turned out to be similar between seeding periods (2-way ANOVA: F2,8 = 3.0829, Results p = 0.0706) and sori groups (2-way ANOVA: F2,8 = 0.1873, p = 0.83). Within each seeding period (Fig. 6a–c), Experiment 1: spore seeding and sori use the spore count decreased significantly over time (chi- squared: χ2 (9) = 10.14, p = 0.0063), whereas the increase Spores settled on all nets during the consecutive seeding in seedling density was only significantly on nets produced periods and for all amount of sori used (Fig. 6a–c). Spores in second (Mann-Whitney two-sample test: p = 0.0030) were present on all counting days, while seedlings (Fig. and third (p = 0.0295) seeding periods. 6d–f) were only present from counting day 19 and on- The additional rope pieces sampled to assess whether set- wards. On counting day 3, the number of spores attached tlement was homogeneous dispersed across the entire net, to the nets was significantly affected by seeding periods (2- showed that this was the case. Both spores and seedlings way ANOVA: F2,8 = 7.2461, p = 0.0049), the second (Fig. showed equal variance between top and bottom of the nets 6b)andthird(Fig.6c) seeding periods (days 3–6 and days (spores: p = 0.17, seedlings: p = 0.40) and means (Mann- 6–9) showing similar and significant higher spore densities Whitney two-sample test: p = 0.40). Furthermore, the densi- (Tukey’s: p = 0.0036) than the first seeding period (days 0– ties showed equal variance horizontally around the net spiral 3) (Fig. 6a). Also, the sori amount used for seeding, sig- (spores: p = 0.23, seedlings: p = 0.33). Hence, the number of nificantly affected the number of attached spores (2-way spores settled on rope pieces cut from the nets was extrapolat- – ANOVA: F2,8 = 4.0989, p = 0.0341), showing significant ed to whole nets (Fig. 7a c) and summed with the number of difference between high and low sori amounts (Tukey’s, detained spores (Fig. 7d–f) to calculate the total number of p = 0.0109). On counting day 19, the number of spores released spores. This number was then used to estimate a net (Fig. 6a–c) and seedlings (Fig. 6d–f)aswellasthespore seeding efficiency of the system (Fig. 7g–i) by Eq. 1. The net Fig. 6 Thenumberofspores(a– c) and seedlings (d–f) attached to the nets and the spore germination percentage (g–i). 4 cm net pieces were counted on days 3, 19, and 32 after each of the three consecutive periods (days 0–3, days 3–6, days 6–9) with the use of different sori amounts (5, 10, 15 g). Data presented as mean ± SE, n =3 106 JApplPhycol Fig. 7 a–i The extrapolated number of spores attached to the nets and d–f the total number of detained spores collected in the spore-detaining tanks (SDTs) as a function of three consecutive seeding periods and the use of three different amounts of sori (5, 10, 15 g FW). g–i Net seeding efficiency, calculated from Eq. 1. Data presented as mean ± SE, n = 3 seeding efficiency of the agitated, flow-through seeding tank F2,8 = 5.07, p = 0.0178) where 15 g sori provided higher system using sori packages was in average 16 ± 1.6% (Fig. yield compared to 5 g sori (Tukey’s: p = 0.0175). In total, 7g–i) and not affected by either seeding period or sori amount the amount of released spores (detained + attached to nets) (2w-an: p > 0.28). This indicates that app. 84% of the released from the three different sori packages during 9 days of tetraspores were washed out and detained (Fig. 7d–f). sporulation was significantly higher for the 15 g sori Effluent spores aggregated as red plumages on the bot- group (1,300,563 ± 71,639), compared to the 10 g sori tom of all the SDTs, and were visible within 3 h during the (959,697 ± 69,618) and the 5 g sori group (522,230 ± first seeding period (days 0–3). The number of detained 61,869) spores. spores from the flow-through system (Fig. 7d–f)wassig- nificantly affected by seeding period (2-way ANOVA: Experiment 2: mixed propagules as seeding inoculum F2,8 =5.66, p = 0.0124), where second period provided higher detained spore yield compared to the third period On counting day 3, the number of reattached spores (Fig. (Tukey’s: p = 0.0117) and similar spore yield as first peri- 8a vs. b) and seedlings (Fig. 8c vs. d) was significantly od (p = 0.6593). The amount of sori used also affected the higher, when the inoculum was dislodged at days 27–29 number of detained spores significantly (2-way ANOVA: compared to days 37–39 (Mann-Whitney two-sample test Fig. 8 Attached spores (a, b)and seedlings (c, d)onropepieces seeded with macerated propagule solution at days 3 and 10 and as a function of age at dislodgement (days 27–29 vs. days 37–39) and agitation period (days 0–2vs. days 10–12). Data is presented as outlier box plots (1st, 3rd quartile whiskers) based on three subpart counts on each of ten pieces of rope (n =10,N =30) 107 JApplPhycol (10), p < 0.0001). Furthermore, the number of reattached system, 9 nets were spore-seeded, with an equivalent linear spores and seedlings on ropes exposed to different agita- length of ~ 126 m of rope, using as little as 5 g FW sori, which tion periods (days 0–2 vs. days 10–12) was significantly result in an average of 9 seedling cm−1 after 32 days. The use different (p < 0.0001, p = 0.0012, respectively). After nurs- of spores for seeding substrates is prospective for larger scale ing these ropes for additional 7 days (counting day 10), the cultivation (Browne 2001;Edwards2007; Werner and Dring effect of agitation period on the density of spores and seed- 2011). The latter argued that an initial seeding density lings remained significant (p = 0.0002, p = 0.0009). In con- of spores cm−1 is required to obtain a final seedling density trast, the effect of inoculum age at dislodgement was in- of ~ 6–8seedlingscm−1 as a mortality rate of spores of 60– significant on the spore density (p = 0.0697) and seedling 80% took place. On top, only male gametophytes developed a density (p =0.9938). thallus, representing 50% of the total amount of spores. The initial spore density encountered in the present study (exp. 1) Experiment 3: including fertilization in hatchery on the nets was lower than 100 spores cm−1; however, we estimated a spore germination success (reciprocal to mortality) After 19 days in petri dish culture, the adjusted seedling num- of 50–80% on the nets similar to what was achieved in a ber was 50.25% higher (Fig. 9a) with male gametes solution previous study (Le Gall et al. 2004). This indicates that the added, compared to the control (two-tailed Student’s t test: seeding and nursery conditions presented here were good, t(10), p < 0.0001), whereas no significant differences were though it might be overestimated, as we were not able to count found at the other days (p > 0.98), except day 3, where the the dying spores. In contrast, we observed a germination suc- control group showed a slightly higher number of seedlings cess of only 8–14% in petri dishes cultured in stagnant sea- (p =0.0042). water under the same conditions (unpublished work), which is The effect of adding male gametes to the rope cultures (Fig. in the range of previous report (Edwards 2007; Edwards and 9b) was significant from day 3 (MANOVA: F1,19 = 121.445, Dring 2011). By using higher amounts of sori in the seeding p < 0.0001) with higher number of seedlings at all counting phase of net, the nets showed higher spore density after 3 days, days (253.2% at day 12). Yet, the effect of time was insignif- but decreased to similar levels for all sori amount used, after icant on the number of seedlings (MANOVA: F3,17 =0.5462, 32 days of nursery, in line with previous findings (Edwards p = 0.6573) but caused a significant interaction term of gamete 2007; Werner and Dring 2011). In present study, the average addition × time: F3,17 = 4.0842, p = 0.0235, because of the seedling density after 32 days of nursery was not significantly higher seedling count after day 0. affected by the sori amounts tested. Besides testing efficient ways to handle sori tissue for optimal spore yield, it is impor- tant that future hatchery trials consider the sori-to-substrate Discussion density, as high spore density seem to even out during nursery; thus, the sori could have been used more efficient. A current The results of this study demonstrate effective methods and hatchery protocol for P. palmata found that a sori-to-substrate − strategies in the pursuit of optimizing the hatchery production ratio of 150 g FW sori 84 m 1 substrate was required to secure of P. palmata by using vertical seeding tanks, consecutive use sufficient seedling density, which converts to ~ 130 kg FW of sori packages and agitation during the seeding phase. The fronds to seed one long-line of 100 m (Werner and Dring strategy of using a secondary seeding inoculum of germinated 2011). In comparison, several kilometers of substrate can be propagules, based on collecting the effluent spores, obviously seeded with motile zoospores of Saccharina latissima by increase the spore use efficiency. With presented seeding using 150 g sori, due to the multifold number of biflagellate Fig. 9 a The normalized seedling count in petri dish cultures (n = 10) of macerated P. palmata propagules as an effect of adding male gametes. b The normalized seedling count on spore-seeded rope cultures (n = 11) as an effect of adding male gametes. Data is presented as outlier box plots (1st, 3rd quartile whiskers) 108 JApplPhycol zoospores released within 1 h and their capability of high The thallus fragments showed a decreasing reattachment dispersal (personal observation). We suggest that a lower probability over time (Fonck et al. 2008). Also, the red sea- amount of sori is sufficient for seeding, while considering weed Gelidium chilense (Montagne) formed bundles of rhi- the sori-to-substrate ratio. zoids in agitated water (Santelices and Varela 1994), while Three times the amount of seeded substrate was pro- absent in Gelidium coulteri cultured in stagnant water condi- duced by using three consecutive seeding periods, com- tions (Macler and West 1987). In comparison, a direct seeding pared to the conventional 3-day seeding phase. The highest method for cultivating the brown macroalgae species seedling density after 32 days for nets seeding was found S. latissima, as a way to optimize hatchery duration and costs, in the second (~ 8 seedlings cm−1 and third seeing period is currently used by the Hortimare Company, as a seeding (~ 10 seedlings cm−1). In the first seeding period (days 0– technique for commercial cultivation in Europe. Here, tiny 3), our tank setup provided a spore-seeding efficiency of germinated and activated sporophytes are applied to substrates 16%, meaning that ~ 84% of the released tetraspores were and establish firm attachment with their developing haptera detained in the down-stream detaining tanks. The amount of organs, with a potential benefit of using a glue (Kerrison et al. detained spores was highest after the second seeding period 2018). Whether the propagule seeding method of P. palmata (days 3–6). Even 38 days after finalizing the last seeding pe- would benefit by using glue as a means to maximize seedling riod, we observed red tetraspore aggregates in some of the density is of high interest due to the high commercial value of spore-detaining tanks. This observation supports previous the biomass. findings, where sori was observed to release for 21 days The inclusion of a fertilization step proved to be highly (Schmedes et al. 2019)to40days(Wood2018). relevant to increase the number of seedlings in the hatchery Several bottlenecks have been identified by using the cur- production, by releasing male gametes and adding this male rent hatchery protocol for P. p alm ata for large-scale gamete solution to the female gametophytes that are develop- cultivation—a protocol where sori is placed above the culti- ing their trichogynes (Mine and Tatewaki 1994;LeGalletal. vation substrates (1:1 areal coverage) in horizontal tanks, 2004). This resulted in at least a doubling of the number of which impose a high sori requirement. Facing other chal- seedlings on spore-seeded rope and suggests that male gam- lenges when hatching rhodophytes, such as seasonal variation etes can be added 10–22 days after seeding substrates to in- in spore availability (Kain 1986;LeGalletal.2004), a relative crease the overall spore use efficiency of P. palmata. low spore release yield (Edwards 2007), poor spore dispersal before settlement (Edwards and Dring 2011), and low survival of spores (Sanderson 2006; Werner and Dring 2011), all im- pose a low spore use efficiency and little control of seedling Conclusions quality. Overall, an even spore dispersal and good seedling density was found in this study using a flow-through system A new method is reported here to handle fertile sori of and relative high aeration, agitating the water. Nevertheless, P. palmata for efficient release and dispersal of tetraspores further investigations of the previous mentioned challenges using sori packages in vertical flow-through tanks. This are highly relevant to optimize before commercial hatcheries achieves a sufficient seedling density ~ 9 seedlings cm−1 can be established. Hence, the recently developed GMA meth- rope. Consecutive use of sori greatly improves the spore od (Schmedes et al. 2019) to improve the spore efficiency for use efficiency and was found to have a positive effect on hatching P. palmata was investigated. This method was ap- seeding quality, even for different sori amounts used in the plied by dislodging and maceration of detained spores, which sporulation phase. The spore inoculation success (net- then germinated into propagules, which again were macerated seeding efficiency ~16%) was relative low; however, the to break aggregates of spores and tiny seedlings into single ~ 84% effluent spores was used in propagule inoculation and small groups of spores and seedlings, before used as a by using the GMA method. Hence, to increase the use of secondary seeding inoculum. The level of water agitation of spores for efficient inoculation, the importance of address- 2.5 L min−1 dispersed the propagules, yet, did not compromise ing following parameters to further improve the spore use the establishment of a discoid reattachment on the ropes (exp. efficiency for seeding P. palmata is needed. The following 2). Results demonstrated that the reattachment was negatively are of importance, i.e., tank typology, water motion for affected by the biological age at dislodgement and amount of optimal spore dispersal and settlement, intermittent batch days in agitation, which adds knowledge to the extent of conditions to increase net seeding efficiency, substrate-to- which macerated P. palmata propagules can be used as volume density, sori position, and flow-through rate. It seeding inoculum. This is in agreement with findings in other was demonstrated that effluent spores from the SDTs were red seaweed species, such as Chondracanthus chamissoi suited as a secondary seeding inoculum, using the (Gigartinales), which forms a substrate reattachment by the germinate-macerate-agitate seeding method (Schmedes production of secondary attachment discs (Sáez et al. 2008). et al. 2019). 109 JApplPhycol Acknowledgments We sincerely thank Pascal David Alain Barreau and extraction of phycobiliproteins and chlorophyll a. Food Res Int 100: Kasper Lenda Andersen for their technical help installing the experimen- 514–521 tal setup, as well as for system maintenance and data collection. Macler BA, West JA (1987) Life history and physiology of the red alga, Gelidium coulteri, in unialgal culture. Aquaculture 61:281–293 Funding information The study was funded by the Joint Doctoral Degree Matos J, Costa S, Rodrigues A, Pereira R, Pinto IS (2006) Experimental agreement between the National Institute of Aquatic Resources (DTU integrated aquaculture of fish and red seaweeds in Northen Portugal. Aqua) at Technical University of Denmark and the Norwegian Aquaculture 252:31–42 University of Science and Technology (NTNU), Norway, as well as the Mine I, Tatewaki M (1994) Attachment and fusion of gametes during Tang.nu project (under Grant Agreement No. 13744, Velux Foundation) fertilization of Palmaria sp. (Rhodophyta). J Phycol 30:55–66 and the MacroSea project, Grant no. 254883, funded by the Research Morgan KC, Simpson FJ (1981) The cultivation of Palmaria palmata: Council of Norway. effect of light intensity and temperature on growth and chemical composition. 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Dublin Kerrison PD, Stanley MS, Hughes AD (2018) Textile substrate seeding of Saccharina latissima sporophytes using a binder: an effective meth- Wood E (2018) Cultivation of Palmaria palmata: spore release, vegeta- od for the aquaculture of kelp. Algal Res 33:352–357 tive propagation and decontamination techniques. Scottish Le Gall L, Pien S, Rusig AM (2004) Cultivation of Palmaria palmata Associaiton for Marine Science (SAMS) and University of the (Palmariales, Rhodophyta) from isolated spores in semi-controlled Highlands and Islands conditions. Aquaculture 229:181–191 Lee D, Mizuho N, Yutaka S, Hiroki S (2017) Anti-inflammatory effects Publisher’snoteSpringer Nature remains neutral with regard to jurisdic- of dulse (Palmaria palmata) resulting from the simultaneous water- tional claims in published maps and institutional affiliations. 110 Paper 4: Growth and productivity in land-based cultivation of P. palmata $IWHUZHHNVRIORZQXWULHQW 012 SXOVHFXOWLYDWLRQ 3$5 3$5 3$5 3$5 3$5 3$5 )ROORZHGE\ZHHNVFXOWLYDWLRQZLWKRQHKLJKQXWULHQWSXOVH 01 111 -RXUQDORI$SSOLHG3K\FRORJ\ 3URGXFWLYLW\DQGJURZWKUDWHLQ3DOPDULDSDOPDWDDIIHFWHGE\VDOLQLW\LUUDGLDQFHDQG QXWULHQWDYDLODELOLW\ದWKHXVHRIQXWULHQWSXOVHVDQGLQWHUYHQWLRQDOFXOWLYDWLRQ 0DQXVFULSW'UDIW 0DQXVFULSW1XPEHU -$3+' )XOO7LWOH 3URGXFWLYLW\DQGJURZWKUDWHLQ3DOPDULDSDOPDWDDIIHFWHGE\VDOLQLW\LUUDGLDQFHDQG QXWULHQWDYDLODELOLW\ದWKHXVHRIQXWULHQWSXOVHVDQGLQWHUYHQWLRQDOFXOWLYDWLRQ $UWLFOH7\SH 2ULJLQDO5HVHDUFK .H\ZRUGV 6HHGVWRFN 5$6VHDZHHG QXWULHQWSXOVHV 1VWDUYDWLRQ 1UHPRYDO EUDFNLVK VDOLQLW\ &RUUHVSRQGLQJ$XWKRU 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