Exploring Nutrient Supplements for Enhanced Growth and Quality of Devaleraea mollis and Palmaria hecatensis

Exploring Nutrient Supplements for Enhanced Growth and Quality of Devaleraea mollis and Palmaria hecatensis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring Nutrient Supplements for Enhanced Growth and Quality of Devaleraea mollis and Palmaria hecatensis Jae Woo Jung, Muriel Dittrich, Jang K. Kim, Schery Umanzor This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4953297/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Devaleraea mollis and Palmaria hecatensis have emerged as potential species for land-based cultivation of red seaweeds in the Pacific Northwest of the United States. Land-based cultivation has the advantage of customization of high-quality biomass production. However, the high material and preparation costs of the von Stosch enrichment medium (VSE) are a limitation of land-based cultivation of D. mollis and P. hecatensis . This study aims to reduce operational and management costs associated with controlling the culturing conditions of D. mollis and P. hecatensis without compromising biomass growth and quality in land-based tank cultivation systems. Five experimental treatments, 1) ambient seawater (AS); 2) VSE; 3) Guillard's f/2 medium (f/2); 4) commercial fertilizer, Jack's Special (JS); 5) JS with vitamin (JSV), were used in the present study. The growth, pigment, and protein content of D. mollis and P. hecatensis were measured. Except for AS, Palmaria hecatensis showed similar growth, pigment, and protein content at all experimental treatments. The growth and protein content of D. mollis exposed to VSE were decreased by nitrogen limitation. However, the protein content of D. mollis exposed to JS and JSV significantly increased without a decrease in growth. Therefore, the commercial fertilizer, Jack's Special (25-5-15), can replace the VSE for D. mollis and P. hecatensis , reducing operational and management costs link to nutrient supplementation. Devaleraea mollis Palmaria hecatensis Nutrient supplements Growth Pigment Protein Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Seaweed aquaculture is one of the fastest-growing maritime industries worldwide (Kim et al. 2019 ; Sultana et al. 2023 ). In 2022, 36.5 million tons of seaweed were produced with a value of 17 billion US $ (FAO FishstatJ database). Most seaweed produced is directed to the food industry, including direct consumption, polysaccharide additives, and functional ingredients (Kim et al. 2017 ; Naylor et al. 2021 ). Hydrocolloids extracted from seaweed stand as the second most valuable segment, while fertilizers and animal feed additives make up the remainder (Barbier et al. 2019 ; Porse and Rudolph 2017 ; Shannon and Abu-Ghannam 2019 ; Naylor et al. 2021 ; Song et al. 2022 ). A global shift towards holistic health and sustainable food sources has recently increased demand for seaweed biomass, positioning it as a staple in functional foods and feeds rich in essential nutrients (Shannon and Abu-Ghannam 2019 ; Yong et al. 2024 ). The Pacific Northwest of the United States is emerging as an optimal hub for the burgeoning seaweed farming industry (Considine et al. 2023 ; Kim et al. 2019 ). While the focus lies on kelp cultivation at sea, other types of seaweed farming are also explored. For example, the red seaweeds Devaleraea mollis (formerly known as Palmaria mollis ) and Palmaria hecatensis have gained interest as potential species for land-based cultivation given their high protein content (Demetropoulos and Langdon 2004a , b ; Kim et al. 2019 ; Saunders et al. 2018 ; Gadberry et al. 2018 ). D. mollis has seen successful growth in outdoor land-based systems across Oregon, California, Hawaii, and more recently indoors in SE Alaska. Cultivation has mainly benefited the abalone industry along the Pacific coast (Evans and Langdon 2000 ; Demetropoulos and Langdon 2004a ; Langdon et al. 2004 ; Evans et al. 2021 ). On the other hand, P. hecatensis represents a novel aquaculture species for Alaska, with indoor land-based cultivation protocols recently developed (Dittrich et al. in prep). Studies on Palmaria palmata , a similar species from the Atlantic, highlight opportunities for researching downstream applications for D. mollis and P. hecatensis beyond abalone farming. (Werner and Dring 2011 ; Corey et al. 2014 ; Rizzo et al. 2024 ). P. palmata is a top candidate for human consumption (Mouritsen et al. 2013 ; Skriptsova and Kalita 2020 ; Skriptsova et al. 2022 ; Stévant et al. 2023 ). It boasts high protein levels ranging from 8–35% of dry weight, contains various essential nutrients, polyunsaturated fatty acids like EPA, and antioxidants, and is reported to have anticancer properties, making it attractive for biomedical research (Morgan et al. 1980 ; Mouritsen et al. 2013 ; Albertos et al. 2019 ; Lopes et al. 2019 ; Foseid et al. 2020 ). Currently, the species is mainly grown on land, with cultivation in Ireland, Canada, Iceland, Norway, and the United States, among others, focusing on a consistent, high-quality supply for the increasing demand in the food and health sectors (Kim et al. 2013 ; Stévant et al. 2023 ) In contrast with farming at sea, one significant advantage of land-based cultivation is the ability to control and optimize seaweed growing conditions. Such controls can facilitate customizing biomass production to ensure high-quality standards and biosafety (Hafting et al. 2012 ; Hafting et al. 2015 ; Barbier et al. 2018 ; Pereira et al. 2024 ). However, fine-tuning environmental factors such as light quality and quantity, temperature, salinity, pH, water turbulence, and nutrient sources plus their ideal concentrations may increase costs, restricting entry and large-scale production (Titlyanov and Titlyanova 2010 ; Kim and Yarish 2014 ; Suthar et al. 2019 ; Araújo et al. 2021 ). Ongoing research aims to develop cost-effective methods to maintain these optimal conditions without compromising biomass quality. Nutrient supplementation can become a significant operational cost at scale (Kim and Yarish 2014 ). For example, von Stosch enrichment medium (VSE), the recommended nutrient medium for indoor cultivation of red seaweeds (Ott 1965 ; Corey et al. 2012 ; Kim et al. 2012 ; Redmond et al. 2014 ), can cost up to $ 20 per liter of solution becoming cost-prohibitive for commercial-scale production (Kim and Yarish 2014 ). Interestingly, despite containing essential nutrients such as nitrate, phosphate, iron, and vitamins, small-scale cultivation trials on D. mollis and P. hecatensis indicate that VSE may not be the most effective enriched medium for the cultivation of these seaweed species (Dittrich et al. in prep). Results were based on qualitative differences observed on thalli grown using VSE, f/2, and Jack's Special (JS) as nutrient sources. Comparable results have been reported for other red seaweed grown in tank conditions. For example, a study on Gracilaria tikvahiae compared the effectiveness of low-cost fertilizers versus VSE as a nutrient source. The findings revealed that JS, a commercially available fertilizer, produced growth rates and productivity comparable to VSE (Kim and Yarish 2014 ). Of relevance, the cost of JS was reported to be 98% lower than VSE, highlighting its economic advantage over reagent-grade nutrient sources. Notably, nitrogen (N) plays a crucial role in seaweed development, with nitrate and ammonium being primary sources in nutrient supplements (Lobban and Harrison 1994 ). The effectiveness of nitrate (NO 3 − ) and ammonium (NH 4 + ) as nitrogen sources is context-dependent and species-specific (Lobban and Harrison 1994 ; Kim et al. 2012 ; Corey et al. 2013 ). For instance, studies conducted on D. mollis co-cultured with abalone showed that nitrate, as a nitrogen source, promoted more growth than ammonium when assessed long-term (9 weeks). However, thalli exposed to ammonium showed more growth in the short term (2–5 weeks) than those exposed to nitrate (Demetropoulos and Langdon 2004b ). Therefore, in this study, we assessed whether different nutrient supplements with nitrogen incorporated as nitrate, ammonium, or both influence growth rates, photosynthesis efficiency, pigment, protein content, and nutrient content in tissue and medium of D. mollis and P. hecatensis . We hypothesized that (i) commercial fertilizers can replace the VSE medium for cultivating both species and (ii) different nitrogen sources will have different effects on growth and overall performance of both species. We seek to reduce operational and management costs associated with controlling culturing conditions of D. mollis and P. hecatensis without compromising biomass growth and quality in land-based tank cultivation systems. 2. Materials and methods 2.1 Sample collection and pre-cultivation Devaleraea mollis and Palmaria hecatensis were collected from Kodiak Island, Alaska. Both species were acclimated and cultured in a seawater flow-through tank in the Mariculture Lab at the Juneau College of Fisheries and Ocean Sciences. Salinity, temperature, and irradiance were maintained at 30 ± 2 ppt, 8℃, and 100 photons m − 2 s − 1 before the experimental period. Guillard's f/2 medium (f/2) was used as a nutrient supplement (Guillard 1975 ). 2.2 Experimental treatments Three different nutrient supplements, 1) von Stosch enrichment medium (VSE; Ott 1965 ); 2) Guillard's f/2 medium (f/2); 3) commercial fertilizer, Jack's Special (25-5-15; JS), were used in the present study. Ambient seawater (AS) was used as a control without a nutrient supplement. Since VSE and f/2 contained vitamins, JS with vitamin (JSV) was also added to the experimental treatments. Thus, five experimental treatments (i.e., S, VSE, f/2, JS, and JSV), were used. Total nitrogen concentrations in f/2, JS, and JSV were adjusted to be the same as those in VSE (500 µM; Table 1 ). The experiment was conducted after seven consecutive days of nitrogen (N) starvation in ambient seawater without any nutrient supplementation. After that, 0.20 g of fresh weight of Devaleraea mollis and Palmaria hecatensis were transferred to independent 250 mL Erlenmeyer flasks filled with an assigned experimental treatment. Each experimental treatment had four replicates. Samples were cultivated in their assigned experimental treatment at 30 ppt, 8 ℃, 100 µmol photons m − 2 s − 1 photosynthetically active radiation (PAR) and 12:12 h (L:D) photoperiod for two weeks. The culturing medium containing the targeted nutrient supplement was renewed every five days to avoid contaminant growth and potential unintended nutrient limitation. The specific growth rate (SGR) of each species was determined using the fresh weight of each sample as a metric. Fresh weight was measured every seven days within the 14 day experimental period. The short experimental period follows our interest in producing the most biomass in the shortest period possible before growth rates of parental thalli decrease (i.e., typically by day 21st after initiating cultures; Dittrich et al. in prep). It is also an adequate timeframe for measuring short-term physiological responses due to the experimental treatments. The specific growth rate of experimental thalli was calculated using the equation as follows (Krzemińska et al. 2014 ): $$\:SGR\left(\%\:{day}^{-1}\right)=\frac{ln{Wt}_{2}-ln{Wt}_{1}}{{t}_{2}-{t}_{1}}\times\:100$$ Where Wt 2 and Wt 1 represent the weights of the thalli on days t 2 and t 1 . Table 1 Micro and macroelement concentration in different nutrient supplement Nutrient (µM) VSE f/2 JS JSV Nitrogen 500 (100% NO 3 - ) 500 (100% NO 3 - ) 500 (57% NO 3 - and 43% NH 4 + ) 500 (57% NO 3 - and 43% NH 4 + ) Phosphorus 30 15 45 45 Iron 1 11.7 0.6 0.6 Manganese 10 0.9 0.3 0.3 EDTA 10 11.7 - - Vitamins Vitamin B 1 and B 12 , Biotin Vitamin B 1 and B 12 , Biotin - Vitamin B 1 and B 12 , Biotin Potassium - - 107 107 Boron - - 0.6 0.6 Copper - 0.04 0.05 0.05 Molybdenum - 0.03 0.03 0.03 Zinc - 0.05 0.004 0.004 Magnesium - - 1.15 1.15 2.3 Nutrient analysis Approximately 50 mg of fresh thalli was collected on day 0 (start) and day 14 (end) of the experiment and dried in a dry oven at 60 ℃ until constant weight. Dried samples were ground to powder using an MM400 Ball Mill (Retsch, Germany). The tissue carbon and nitrogen content of each sample was analyzed using a CHN analyzer (Thermo Fisher, USA). Moreover, water samples from each nutrient supplement stock and experimental treatment were collected and filtered through 0.45 µm syringed filters (33mm diameter, Chromdisc, Korea) when the media was renewed. The concentration of nitrate (nitrite), ammonium, and phosphorus was measured using a Continuous Segmented Flow Analyzer (QuAAtro 39, SEAL Analytical). 2.4 Photosynthesis efficiency and chlorophyll-a Photosynthesis efficiency and chlorophyll-a content were measured on day 0 and day 14 of the experiment. The minimum fluorescence (Fo) and maximum fluorescence (Fm) were measured by pulse amplitude modulated fluorometry (Junior-PAM; Walz, Germany). The maximum quantum yield of PS Ⅱ (Fv/Fm) was calculated as Fv/Fm = (Fm-Fo)/Fm. Chlorophyll-a content was determined using an extract prepared with approximately 20 mg of fresh thalli placed in 2 mL of ice-cold methanol (95%) and kept at 4 ℃ for 24h at dark. Light absorbance of the extract was measured at 666 and 653 nm and calculated as mg g − 1 fresh weight. 2.5 Total protein and phycobiliprotein Total protein content was measured according to the Bradford protein assay at day 0 and day 14 of the experiment (Bradford 1976 ). Approximately 50 mg of fresh thalli were homogenized with 1 mL of potassium phosphate buffer (50mM, pH7) containing 0.25% Triton X-100 and 1% polyvinylpyrrolidone in cold. The homogenates were centrifuged at 12000 g for 10 minutes at 4 ℃. Bradford's reagent (1 mL) was then added to 100 µL of supernatant and incubated at room temperature for 5 minutes. Absorbance was measured at 595 nm within 1 hour. Total protein contents were calculated as mg g − 1 fresh weight. Bovine serum albumin was used as a standard. Phycobiliproteins (phycoerythrin and phycocyanin) were also measured on day 0 and day 14 of the experiment (Beer and Eshel 1985 ). Approximately 50 mg of fresh thalli was ground up with 2.5 mL sodium phosphate buffer (0.1M, pH6.5) and kept at 4 ℃ for 24 hours in darkness. The mixture was centrifuged at 19000g for 20 minutes. The supernatant was measured at 455, 564, 592, 618, and 645 nm and expressed as mg g − 1 fresh weight. 2.6 Statistical analysis One-way ANOVA and Tukey's HSD test (p < 0.05) were conducted to check for statistical differences among nutrient supplements at day 0 and day 14 of the experiment. A t-test was used to detect significant differences between day 0 and day 14 of the experiment within samples exposed to the same nutrient supplement. All data were checked for normality using the Kolmogorov-Smirnov test and homogeneity of variance using Levene's test. Data were analyzed using the statistical software SPSS 25.0. 3. Results The specific growth rate (SGR) of Devaleraea mollis on day 7 and day 14 showed significant differences due to the nutrient supplements (p < 0.001 and p = 0.040, respectively). At day 7, the SGR of D. mollis exposed to von Stosch enrichment medium (VSE) and Guillard's f/2 medium (f/2) were significantly higher than those exposed to ambient seawater (AS), Jack's Special (JS), and JS with vitamin (JSV) (Fig. 1 A; p < 0.05). At day 14, differences were neglectable, with the only significant difference detected between SGRs of samples exposed to f/2 versus AS (Fig. 1 A; p < 0.05). Moreover, results show a significant decrease in SGR of samples exposed to AS and VSE between day 7 and day 14 (Fig. 1 A; p = 0.040 and p = 0.034, respectively). On the other hand, the SGR of Palmaria hecatensis was not affected by the experimental treatments (Fig. 1 B; p > 0.05). Through the experiment, D. mollis showed a faster growth rate than P. hecatensis , with SGR of the former more than 300% of the latter. Although results varied, the experimental treatments also influenced tissue nitrogen (N) content and carbon:nitrogen (C:N) ratio for both species. The tissue nitrogen (N) content of D. mollis exposed to f/2, JS, and JSV was significantly higher than those growing in AS and VSE (Fig. 2 B; p < 0.001. The tissue C:N ratio followed the opposite trend, with thalli exposed to AS and VSE showing significantly higher than those in f/2, JS, and JSV (Fig. 2 C; p < 0.001). On the other hand, tissue nitrogen content of P. hecatensis showed significant differences only between JSV and AS, with JSV showing higher values (Fig. 2 E; p = 0.019). The tissue C:N ratio of P. hecatensis exposed to JS and JSV was significantly lower than thalli exposed to AS (Fig. 2 F; p = 0.014 and p 0.05). By days 0 and 14, mean dissolved inorganic nitrogen (DIN) and phosphate (DIP) uptake rates (µM day 7⁻¹) of D. mollis exposed to VSE were significantly higher than those exposed to f/2, JS, and JSV (Table 2 ). Conversely, the mean DIN uptake rates of P. hecatensis exposed to VSE were significantly lower than those of thalli exposed to f/2, JS, and JSV at both sampling periods (Table 3 ). For D. mollis , the mean DIN:DIP uptake ratio was 16:1 (VSE), 25:1 (f/2), and 23:1 (JS and JSV) by day 7. These ratios showed slight variation by day 14 of the experiment, with values of 15:1 (VSE), 26:1 (f/2), 20:1, and 21:1 (JSV) (Table 2 ). For P. hecatensis , the mean DIN:DIP uptake ratio was 12:1 (VSE), 15:1 (f/2), 14:1 (JS and JSV) by day 7. These ratios also showed slight variation by day 14 of the experiment, with values of 11:1 (VSE), 15:1 (f/2) 12:1 and 13:1 (JSV) (Table 3 ). Table 2 Dissolved inorganic nitrogen (DIN) and phosphate (DIP) uptake and their ratio of Devaleraea mollis exposed to ambient seawater (AS), von Stosch enrichment medium (VSE), Guillard's f/2 medium (f/2), Jack's Special (JS), and JS with vitamin (JSV). Letters represent significant differences among nutrient supplement treatments. Nutrient uptake (µM day 7 − 1 ) VSE f/2 JS JSV Day 7 DIN 490.74 ± 0.02 c 432.72 ± 5.75 a 459.62 ± 2.27 b 461.47 ± 3.17 b Nitrate (NO 3 − ) 490.74 ± 0.02 432.72 ± 5.75 260.25 ± 1.94 259.18 ± 3.29 Ammonium (NH 4 + ) - - 199.36 ± 3.15 205.21 ± 6.64 DIP 31.07 ± 0.26 b 17.17 ± 0.13 a 21.02 ± 4.66 a 21.13 ± 3.13 a N : P ratio 16:1 25:1 23:1 23:1 Day 14 DIN 490.76 ± 0.02 c 433.24 ± 6.55 a 464.39 ± 8.42 b 461.95 ± 2.43 b Nitrate (NO 3 − ) 490.76 ± 0.02 433.24 ± 6.55 260.79 ± 2.46 258.91 ± 3.62 Ammonium (NH 4 + ) - - 200.67 ± 3.3 203.04 ± 4.32 DIP 32.63 ± 0.26 c 16.96 ± 0.25 a 23.05 ± 3.58 b 21.79 ± 2.58 b N : P ratio 15:1 26:1 20:1 21:1 Table 3 Dissolved inorganic nitrogen (DIN) and phosphate (DIP) uptake and ratio of Palmaria hecatensis at ambient seawater (AS), von Stosch enrichment medium (VSE), Guillard's f/2 medium (f/2), Jack's Special (JS) and JS with vitamin (JSV). Different letters represent the significant difference among nutrient supplements. Nutrient uptake (µM day 7 − 1 ) VSE f/2 JS JSV Day 7 DIN 45.88 ± 7.92 a 66.87 ± 2.71 b 62.54 ± 5.74 b 58.70 ± 1.70 b Nitrate (NO 3 − ) 45.88 ± 7.92 66.87 ± 2.71 48.64 ± 6.70 45.58 ± 0.47 Ammonium (NH 4 + ) - - 13.90 ± 1.11 13.12 ± 1.83 DIP 4.08 ± 0.55 4.60 ± 0.34 4.59 ± 0.09 4.34 ± 0.09 N : P ratio 12:1 15:1 14:1 14:1 Day 14 DIN 47.23 ± 8.86 a 69.18 ± 2.93 b 57.15 ± 8.25 b 60.98 ± 4.71 b Nitrate (NO 3 − ) 47.23 ± 8.86 69.18 ± 2.93 44.20 ± 8.25 47.44 ± 3.16 Ammonium (NH 4 + ) - - 12.95 ± 1.30 13.54 ± 1.92 DIP 4.16 ± 0.18 4.58 ± 0.46 4.74 ± 0.29 4.73 ± 0.12 N : P ratio 11:1 15:1 12:1 13:1 The maximum quantum yield of PS Ⅱ (Fv/Fm) of D. mollis and P. hecatensis was not affected by the experimental nutrient supplements (Fig. 3 ; p > 0.05). Conversely, results show a significant difference in chlorophyll-a content due to the experimental treatments in D. mollis and P. hecatensis (p < 0.001). The chlorophyll-a content in D. mollis exposed to f/2, JS, and JSV were significantly higher than those of thalli exposed to AS at day 14 of the experiment (Fig. 4 A; p < 0.05). Moreover, chlorophyll-a content of D. mollis exposed to f/2, JS, and JSV was significantly higher on day 14 of the experiment than on day 0 (Fig. 4 A; p < 0.05). On the other hand, chlorophyll-a content of P. hecatensis exposed to VSE, f/2, JS, and JSV were also significantly higher than those thalli exposed to AS at day 14 of the experiment (Fig. 4 B; p < 0.001). On day 14 of the experiment, chlorophyll-a content of P. hecatensis exposed to AS was significantly lower than that at day 0 of the experiment (Fig. 4 B; p = 0.005). No significant difference in chlorophyll-a between day 0 and day 14 of the experiment was detected for any other treatments. Similarly, nutrient supplement treatments significantly influenced the total protein content of D. mollis and P. hecatensis (p < 0.001). The total protein content of D. mollis exposed to f/2, JS, and JSV were significantly higher than those at AS and VSE by day 14 (Fig. 5 A; p < 0.01). Results show a significant decrease in the total protein content of D. mollis exposed AS and VSE between day 0 and day 14 of the experiment (Fig. 5 A; p < 0.001). In contrast, samples exposed to JS and JSV showed a significant increase on day 14 compared to day 0 of the experiment (Fig. 5 A; p < 0.05). P. hecatensis also showed significantly different responses, with samples exposed to VSE, f/2, JS, and JSV having a higher protein content than those exposed to AS (Fig. 5 B; p < 0.05). All treatments, except for AS, showed significantly higher protein content at day 14 versus day 0 of the experiment (Fig. 5 B; p < 0.05). In contrast, with protein content, the phycobiliprotein content (phycocyanin and phycoerythrin) of D. mollis and P. hecatensis followed different trends. Phycobiliprotein content in D. mollis was significantly affected by the experimental treatment (p 0.05). Specifically, phycocyanin and phycoerythrin in D. mollis exposed to f/2, JS, and JSV were significantly higher than those exposed to AS and VSE by day 14 (Fig. 6 A and B; p < 0.01). On day 14 of the experiment, phycocyanin and phycoerythrin of D. mollis exposed to AS were significantly lower, whereas thalli exposed to f/2, JS, and JSV were significantly higher by day 14 compared to day 0 (Fig. 6 A and B; p < 0.05). 4. Discussion On-land tank seaweed cultivation offers numerous benefits, including improved control over biomass quality. This cultivation technique has demonstrated the highest yields per square meter of water surface compared to other methods (Titlyanov and Titlyanova 2010 ; Hwang et al. 2020 ; Shin et al. 2020 ). In this study, we investigated whether the cost of tank cultivation could be reduced without compromising the growth and performance of the red seaweeds Devalaraea mollis and Palmaria hecatensis by using commercial-grade nutrient supplementation instead of the recommended reagent-grade nutrient source. Our results showed that the commercial fertilizer Jack's Special (JS) effectively replaced the von Stosch enrichment medium (VSE), favoring growth and protein content of both species. As expected, there were differences in crop performance based on nutrient source and species over time. These findings align with previous cultivation trials where the reagent-grade product, VSE, showed limited benefits for the growth performance of D. mollis and P. hecatensis (Dittrich et al. in prep). In this study, we focused on nitrogen, an essential element for seaweed growth and often a limiting factor (Kim et al. 2007 ; Hurd et al. 2014 ; Roleda and Hurd 2019 ; Xu et al. 2020 ; Bao et al. 2023 ). Our results revealed that the Specific Growth Rate (SGR) of D. mollis exposed to ambient seawater (AS) and VSE significantly decreased from day 7 to day 14. The carbon to nitrogen ratio (C:N), a key indicator of nitrogen status (Kim et al. 2007 ; Hurd et al. 2014 ), showed nitrogen limitation at day 14 of the experiment, with mean tissue C:N of 16.2 ± 1.77 and 15.4 ± 1.40 for thalli growing with AS and VSE, respectively. In contrast, the C:N of samples exposed to Guillard's f/2 medium (f/2), JS, and JS with vitamins (JSV) indicated nitrogen accumulation (9.09 ± 1.06, 8.54 ± 1.07, and 7.60 ± 0.56, respectively). This reduction in growth linked to nitrogen limitation aligns with studies conducted on Pyropia yezoensis , Gracilariopsis lemaneiformis , Gracilaria tikvahiae and Ulva pertusa (Liu and Dong 2001 ; Kim et al. 2007 ; Kim and Yarish 2014 ; Li et al. 2019 ; Liu et al. 2019a ; Liu et al. 2019b ). The SGR of P. hecatensis was not affected by the type of nutrient supplementation. However, similar to D. mollis , the mean tissue C:N of thalli exposed to JS and JSV indicated nitrogen accumulation (9.96 ± 0.30 and 8.89 ± 0.19, respectively), which was significantly lower than in P. hecatensis grown in AS. Under limiting conditions, seaweed obtain nitrogen through the catabolism of nitrogen-based compounds, leading to reduced concentrations of these compounds, such as chlorophyll-a, phycobiliproteins, and proteins (Kim et al. 2008 ; Roleda and Hurd 2019 ; Chen et al. 2023 ). The catabolism of nitrogen-based compounds might maintain or increase growth in the short term, but ultimately, it might inhibit growth in the long term if the limitation persists. Overall, our results indicate that JS and modified JS with vitamins can replace VSE and f/2 for growing D. mollis and P. hecatensis , with JS and JSV showing the highest protein content in D. mollis . VSE and f/2 contain only nitrate as a nitrogen source, while JS and JSV contain both nitrate and ammonium (Kim and Yarish 2014 ). Ammonium contributed to higher protein content due to its direct incorporation into amino acids (Lobban and Harrison 1994 ; Kim et al. 2012 ). On the other hand, although the targeted seaweed could have incorporated nitrate, the process was not conducive to promoting the most growth, as it required reduction for assimilation (Hurd et al. 1995 ; Pritchard et al. 2015 ). First, nitrate reductase (NR) converts nitrate to nitrite (NO 2 - ), and nitrite reductase (NiR) converts nitrite to ammonium. These processes require energy in the form of NADPH or ferredoxin, making the assimilation of nitrate more energetically costly compared to ammonium (Jin et al. 1998 ; Cohen and Fong 2004 ). Additionally, although temperature was not an experimental factor in this study, it could have affected the enzymatic activity and active transport of nitrate, but not passive diffusion of ammonium (Nishihara et al. 2005 ; Roleda and Hurd 2019 ). In this case, ammonium transporters facilitate the rapid influx of ammonium ions into the cells, making it easier for the seaweed to utilize ammonium when available. Our results show that the phycobiliprotein content of D. mollis in JS and JSV was significantly higher than in AS. This finding aligns with the notion that seaweed can store excess nitrogen through pigments and other compounds, supporting future growth under nitrogen depletion (McGlathery et al. 1996 ; Naldi and Wheeler 1999 ). For instance, Grateloupia turuturu stores extra ammonium in phycobiliprotein (Chen et al. 2023 ), while Palmaria palmata can synthesize and accumulate phycoerythrin under nutrient-replete conditions (Furuta et al. 2016 ; Schmedes and Nielsen 2020 ; Vasconcelos et al. 2022 ). Here, D. mollis showed protein profiles akin to those of Palmaria palmata . Although the protein content of P. hecatensis exposed to VSE, f/2, JS, and JSV was significantly higher than in AS, there were no differences in phycobiliprotein content among nutrient supplements. It has been documented that nitrogen storage pools can vary between species (Naldi and Wheeler 1999 ; Young et al. 2007 ; Chen et al. 2023 ; Mendez and Kwon 2023 ), which seems to be the case here. P. hecatensis may likely require tailored nutrient management strategies to optimize their growth and protein content, highlighting the need for species-specific cultivation practices in on-land tank systems. Finally, our results emphasize the need to consider the unique nutritional and storage requirements of targeted seaweed species to enhance productivity and quality. Further research is necessary to understand the specific nitrogen storage mechanisms and protein compositions of P. hecatensis , which can lead to more effective fertilizers and growth media. These insights can inform species selection based on their growth characteristics and nutrient requirements for commercial seaweed production, potentially improving yield and reducing costs. Additionally, understanding species-specific responses to nutrient supplementation can inform ecological studies in the context of changing environmental conditions. Furthermore, the knowledge of distinct nitrogen storage pools and protein compositions can be leveraged in biotechnological applications, such as producing bioactive compounds, pigments, and other valuable products from red seaweed. Lastly, trace metals such as zinc and copper, essential for photosynthesis, growth, and cellular metabolism, also play a crucial role in seaweed cultivation (Howarth and Cole 1985 ; Demetropoulos and Langdon 2004b ). Further studies on the effect of different trace metal concentrations among nutrient supplements would add to the increasing literature regarding the cultivation of red seaweeds. 5. Conclusion In conclusion, D. mollis showed decreased growth when cultured with VSE due to nitrogen limitation, indicated by lower total protein content and higher tissue C:N ratio. The highest protein content was observed with the commercial fertilizer Jack's Special (25-5-15), likely due to the presence of ammonium. P. hecatensis demonstrated similar growth, pigment, and protein content across all nutrient supplements, indicating that Jack's Special can effectively replace VSE for both species, reducing operational and management costs. Declarations Ethical Approval Not Applicable Funding The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Availability of data and materials Data will be made available upon request to the authors. Author Contribution JW J conceptualized, investigated, conducted formal analysis, wrote the original draft, and reviewed and edited the manuscript. MD conceptualized, reviewed, and edited the manuscript. JK K and SU conceptualized, conducted formal analysis, reviewed and edited the manuscript, acquired funding, and supervised. Acknowledgement This study was funded by the Ministry of Education (NRF-2017R1A6A1A06015181), the Ministry of Science and ICT through the National Research Foundation of Korea (NRF-2021K1A3A1A05086015), the Ministry of Oceans and Fisheries of Korea (Project No. 20190518) and the State of Alaska (Project No. G00015020). References Albertos I, Martin-Diana A, Burón M, Rico D (2019) Development of functional bio-based seaweed (Himanthalia elongata and Palmaria palmata) edible films for extending the shelflife of fresh fish burgers. Food Packaging and Shelf Life 22:100382 Andrade LR, Farina M, Amado Filho GM (2004) Effects of copper on Enteromorpha flexuosa (Chlorophyta) in vitro. Ecotoxicology and environmental safety 58 (1):117-125 Araújo R, Vázquez Calderón F, Sánchez López J, Azevedo IC, Bruhn A, Fluch S, Garcia Tasende M, Ghaderiardakani F, Ilmjärv T, Laurans M (2021) Current status of the algae production industry in Europe: an emerging sector of the blue bioeconomy. Frontiers in Marine Science 7:626389 Bao M, Xing Q, Park J-S, He P, Zhang J, Yarish C, Kim JK (2023) Temperature and high nutrients enhance hypo-salinity tolerance of the bloom forming green alga, Ulva prolifera. Harmful Algae 123:102402 Barbier M, Charrier B, Araujo R, Holdt S, Jacquemin B, Rebours C (2019) Phycomorph European guidelines for a sustainable aquaculture of seaweeds. COST action FA1406 doi org/1021411/2c3w-yc73 Barbier M, Charrier B, Araujo R, Holdt S, Jacquemin B, Rebours C, Abreu H, Bruhn A, De Clerck O, Funderund J (2018) PEGASUS: Phycomorph european guidelines for sustainable aquaculture of seaweeds. Centro Oceanográfico de Santander Beer S, Eshel A (1985) Determining phycoerythrin and phycocyanin concentrations in aqueous crude extracts of red algae. Aust J Mar Freshw Res 36 (6):785-792 Bothe H (2011) Plants in heavy metal soils. Detoxification of heavy metals:35-57 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry 72 (1-2):248-254 Chen Y, Lan L, Zhang J, Wang Q, Liu Y, Li H, Gong Q, Gao X (2023) Physiological Impacts of Nitrogen Starvation and Subsequent Recovery on the Red Seaweed Grateloupia turuturu (Halymeniaceae, Rhodophyta). Sustainability 15 (9):7032 Cohen RA, Fong P (2004) Nitrogen uptake and assimilation in Enteromorpha intestinalis (L.) Link (Chlorophyta): using 15N to determine preference during simultaneous pulses of nitrate and ammonium. Journal of experimental marine biology and ecology 309 (1):67-77 Coleman JE (1998) Zinc enzymes. Current opinion in chemical biology 2 (2):222-234 Considine ME, Kirchner G, Carter J, Waters T (2023) Situation analysis for Oregon's emergent seaweed aquaculture industry. Corey P, Kim J, Garbary D, Prithiviraj B, Duston J (2012) Bioremediation potential of Chondrus crispus (Basin Head) and Palmaria palmata: effect of temperature and high nitrate on nutrient removal. Journal of Applied Phycology 24:441-448 Corey P, Kim J, Duston J, Garbary D (2014) Growth and nutrient uptake by Palmaria palmata (Palmariales, Rhodophyta) integrated with Atlantic halibut recirculating aquaculture. Algae 29:35-45 Corey P, Kim JK, Duston J, Garbary DJ, Prithiviraj B (2013) Bioremediation potential of Palmaria palmata and Chondrus crispus (Basin Head): effect of nitrate and ammonium ratio as nitrogen source on nutrient removal. Journal of applied phycology 25:1349-1358 Demetropoulos CL, Langdon CJ (2004a) Effects of nutrient enrichment and biochemical composition of diets of Palmaria mollis on growth and condition of Japanese abalone, Haliotis discus hannai and red abalone, Haliotis rufescens. Journal of experimental marine biology and ecology 308 (2):185-206 Demetropoulos CL, Langdon CJ (2004b) Enhanced production of Pacific dulse (Palmaria mollis) for co-culture with abalone in a land-based system: nitrogen, phosphorus, and trace metal nutrition. Aquaculture 235 (1-4):433-455 Evans F, Langdon CJ (2000) Co-culture of dulse Palmaria mollis and red abalone Haliotis rufescens under limited flow conditions. Aquaculture 185 (1-2):137-158 Evans S, Rorrer GL, Langdon CJ (2021) Cultivation of the macrophytic red alga Palmaria mollis (Pacific dulse) on vertical arrays of mesh panels in aerated tanks. Journal of Applied Phycology 33 (6):3915-3926. doi:10.1007/s10811-021-02582-1 Foseid L, Natvik I, Devle H, Ekeberg D (2020) Identification of fatty acids in fractionated lipid extracts from Palmaria palmata, Alaria esculenta and Saccharina latissima by off-line SPE GC-MS. Journal of Applied Phycology 32 (6):4251-4262 Fredriksen S, Rueness J (1989) Culture studies of Gelidium latifolium (Grev.) Born et Thur.(Rhodophyta) from Norway. Growth and nitrogen storage in response to varying photon flux density, temperature and nitrogen availability. Furuta T, Miyabe Y, Yasui H, Kinoshita Y, Kishimura H (2016) Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata. Marine Drugs 14 (2):32 Gadberry BA, Colt J, Maynard D, Boratyn DC, Webb K, Johnson RB, Saunders GW, Boyer RH, Gadberry BA, Colt J (2018) Intensive land-based production of red and green macroalgae for human consumption in the Pacific Northwest: an evaluation of seasonal growth, yield, nutritional composition, and contaminant levels. Algae 33 (1):109-125 Guillard RR Culture of phytoplankton for feeding marine invertebrates. In: Culture of marine invertebrate animals: proceedings—1st conference on culture of marine invertebrate animals greenport, 1975. Springer, pp 29-60 Hafting JT, Craigie JS, Stengel DB, Loureiro RR, Buschmann AH, Yarish C, Edwards MD, Critchley AT (2015) Prospects and challenges for industrial production of seaweed bioactives. Journal of Phycology 51 (5):821-837 Hafting JT, Critchley AT, Cornish ML, Hubley SA, Archibald AF (2012) On-land cultivation of functional seaweed products for human usage. Journal of Applied Phycology 24:385-392 Hecky R, Kilham P (1988) Nutrient limitation of phytoplankton in freshwater and marine environments: a review of recent evidence on the effects of enrichment 1. Limnology and oceanography 33 (4part2):796-822 Howarth RW, Cole JJ (1985) Molybdenum availability, nitrogen limitation, and phytoplankton growth in natural waters. Science 229 (4714):653-655 Hurd CL, Berges JA, Osborne J, Harrison PJ (1995) An in Vitro Nitrate Reductase Assay for Marine Macroalgae: Optimization and Characterization of the Enzyme for Fucus Gardneri (PHAEOPHYTA) 1. Journal of Phycology 31 (5):835-843 Hurd CL, Harrison PJ, Bischof K, Lobban CS (2014) Seaweed ecology and physiology. Cambridge University Press Hwang EK, Choi HG, Kim JK (2020) Seaweed resources of Korea. Botanica Marina 63 (4):395-405 Jin T, Huppe HC, Turpin DH (1998) In vitro reconstitution of electron transport from glucose-6-phosphate and NADPH to nitrite. Plant physiology 117 (1):303-309 Kim J, Stekoll M, Yarish C (2019) Opportunities, challenges and future directions of open-water seaweed aquaculture in the United States. Phycologia 58 (5):446-461 Kim JK, Duston J, Corey P, Garbary DJ (2013) Marine finfish effluent bioremediation: effects of stocking density and temperature on nitrogen removal capacity of Chondrus crispus and Palmaria palmata (Rhodophyta). Aquaculture 414:210-216 Kim JK, Kraemer GP, Neefus CD, Chung IK, Yarish C (2007) Effects of temperature and ammonium on growth, pigment production and nitrogen uptake by four species of Porphyra (Bangiales, Rhodophyta) native to the New England coast. Journal of Applied Phycology 19:431-440 Kim JK, Kraemer GP, Yarish C (2008) Physiological activity of Porphyra in relation to eulittoral zonation. Journal of Experimental Marine Biology and Ecology 365 (2):75-85 Kim JK, Kraemer GP, Yarish C (2012) Metabolic plasticity of nitrogen assimilation by Porphyra umbilicalis (Linnaeus) Kützing. Journal of Ocean University of China 11 (4):517-526. doi:10.1007/s11802-012-2116-2 Kim JK, Yarish C (2014) Development of a sustainable land-based Gracilaria cultivation system. Algae 29 (3):217-225 Kim JK, Yarish C, Hwang EK, Park M, Kim Y, Kim JK, Yarish C, Hwang EK, Park M, Kim Y (2017) Seaweed aquaculture: cultivation technologies, challenges and its ecosystem services. Algae 32 (1):1-13 Krzemińska I, Pawlik-Skowrońska B, Trzcińska M, Tys J (2014) Influence of photoperiods on the growth rate and biomass productivity of green microalgae. Bioprocess and biosystems engineering 37:735-741 Langdon C, Evans F, Demetropoulos C (2004) An environmentally-sustainable, integrated, co-culture system for dulse and abalone production. Aquacultural engineering 32 (1):43-56 Li C, Ariga I, Mikami K (2019) Difference in nitrogen starvation-inducible expression patterns among phylogenetically diverse ammonium transporter genes in the red seaweed Pyropia yezoensis. American Journal of Plant Sciences 10 (8):1325-1349 Li YX, Zhou S, Zhao FJ, Liu Y, Fan PP, Wang GC (2010) Physiological responses of Porphyra haitanesis to different copper and zinc concentrations. Brazilian Journal of Oceanography 58:261-267 Liu J-W, Dong S-L (2001) Comparative studies on utilizing nitrogen capacity between two macroalgae Gracilaria tenuistipitata var. liui (Rhodophyta) and Ulva pertusa (Chlorophyta) I. Nitrogen storage under nitrogen enrichment and starvation. Journal of Environmental Sciences 13 (3):318-322 Liu X, Wen J, Chen W, Du H (2019a) Physiological effects of nitrogen deficiency and recovery on the macroalga Gracilariopsis lemaneiformis (Rhodophyta). Journal of Phycology 55 (4):830-839 Liu X, Wen J, Zheng C, Jia H, Chen W, Du H (2019b) The impact of nitrogen deficiency and subsequent recovery on the photosynthetic performance of the red macroalga Gracilariopsis lemaneiformis. Journal of Applied Phycology 31:2699-2707 Lobban CS, Harrison PJ (1994) Seaweed ecology and physiology. Cambridge University Press, Lopes D, Melo T, Meneses J, Abreu MH, Pereira R, Domingues P, Lillebø AI, Calado R, Domingues MR (2019) A new look for the red macroalga Palmaria palmata: A seafood with polar lipids rich in EPA and with antioxidant properties. Marine drugs 17 (9):533 Mamun M, Kwon S, Kim J-E, An K-G (2020) Evaluation of algal chlorophyll and nutrient relations and the N:P ratios along with trophic status and light regime in 60 Korea reservoirs. Science of The Total Environment 741:140451. doi:https://doi.org/10.1016/j.scitotenv.2020.140451 McGlathery KJ, Pedersen MF, Borum J (1996) Changes in intracellular nitrogen pools and feedback controls on nitrogen uptake in Chaetomorpha linum (Chlorophyta) 1. Journal of Phycology 32 (3):393-401 Mendez RL, Kwon JY (2023) Precipitation and characterization of Pacific dulse (Devaleraea mollis) proteins. Journal of Applied Phycology 35 (1):301-315 Morgan KC, Wright JL, Simpson F (1980) Review of chemical constituents of the red alga Palmaria palmata (dulse). Economic Botany 34 (1):27-50 Mouritsen OG, Dawczynski C, Duelund L, Jahreis G, Vetter W, Schröder M (2013) On the human consumption of the red seaweed dulse (Palmaria palmata (L.) Weber & Mohr). Journal of Applied Phycology 25:1777-1791 Naldi M, Wheeler PA (1999) Changes in nitrogen pools in Ulva fenestrata (Chlorophyta) and Gracilaria pacifica (Rhodophyta) under nitrate and ammonium enrichment. Journal of Phycology 35 (1):70-77 Naylor RL, Hardy RW, Buschmann AH, Bush SR, Cao L, Klinger DH, Little DC, Lubchenco J, Shumway SE, Troell M (2021) A 20-year retrospective review of global aquaculture. Nature 591 (7851):551-563 Nishihara GN, Terada R, Noro T (2005) Effect of temperature and irradiance on the uptake of ammonium and nitrate by Laurencia brongniartii (Rhodophyta, Ceramiales). Journal of Applied Phycology 17:371-377 Ott FD (1965) Synthetic media and techniques for the xenic cultivation of marine algae and flagellate. Va J Sci 16:205-218 Pereira R, Yarish C, Critchley AT (2024) Seaweed aquaculture for human foods in land based and IMTA systems. In: Applications of Seaweeds in Food and Nutrition. Elsevier, pp 77-99 Porse H, Rudolph B (2017) The seaweed hydrocolloid industry: 2016 updates, requirements, and outlook. Journal of applied phycology 29 (5):2187-2200 Pritchard DW, Hurd CL, Beardall J, Hepburn CD (2015) Restricted use of nitrate and a strong preference for ammonium reflects the nitrogen ecophysiology of a light‐limited red alga. Journal of phycology 51 (2):277-287 Redmond S, Green L, Yarish C, Kim J, Neefus C (2014) New England seaweed culture handbook: nursery systems. Rizzo NR, Beckert SB, Boles SE, Gross JA (2024) Temperature-induced variations in dulse (Devaleraea mollis) nutrition provide indirect benefits on juvenile red abalone (Haliotis rufescens) Growth. Frontiers in Marine Science 11. doi:10.3389/fmars.2024.1336793 Roleda MY, Hurd CL (2019) Seaweed nutrient physiology: application of concepts to aquaculture and bioremediation. Phycologia 58 (5):552-562. doi:10.1080/00318884.2019.1622920 Saunders GW, Jackson C, Salomaki ED (2018) Phylogenetic analyses of transcriptome data resolve familial assignments for genera of the red-algal Acrochaetiales-Palmariales Complex (Nemaliophycidae). Molecular phylogenetics and evolution 119:151-159 Schmedes PS, Nielsen MM (2020) Productivity and growth rate in Palmaria palmata affected by salinity, irradiance, and nutrient availability—the use of nutrient pulses and interventional cultivation. Journal of Applied Phycology 32 (6):4099-4111 Shannon E, Abu-Ghannam N (2019) Seaweeds as nutraceuticals for health and nutrition. Phycologia 58 (5):563-577. doi:10.1080/00318884.2019.1640533 Shin SK, Kim S-K, Kim J-H, Han T, Yarish C, Kim JK (2020) Effects of stocking density on the productivity and nutrient removal of Agarophyton vermiculophyllum in Paralichthys olivaceus biofloc effluent. Journal of Applied Phycology 32:2605-2614 Singh S, Parihar P, Singh R, Singh VP, Prasad SM (2016) Heavy metal tolerance in plants: role of transcriptomics, proteomics, metabolomics, and ionomics. Frontiers in plant science 6:165395 Skriptsova A, Kalita T (2020) A re-evaluation of Palmaria (Palmariaceae, Rhodophyta) in the Northwest Pacific. European Journal of Phycology 55 (3):266-274 Skriptsova AV, Suzuki M, Semenchenko AA (2022) Morphological variation in Northwest Pacific Devaleraea mollis and description of D. inkyuleei sp. nov.(Palmariaceae, Rhodophyta). Phycologia 61 (6):606-615 Smith VH, Schindler DW (2009) Eutrophication science: where do we go from here? Trends in ecology & evolution 24 (4):201-207 Song H-I, Han S, Park J-S, Kim Y-J, Jeong C-B, Yarish C, Kim JK (2022) Dark treatment effect on the carrageenan characterization in a red alga, Chondrus crispus. Algal Research 68:102889 Stévant P, Schmedes PS, Le Gall L, Wegeberg S, Dumay J, Rebours C (2023) Concise review of the red macroalga dulse, Palmaria palmata (L.) Weber & Mohr. Journal of Applied Phycology 35 (2):523-550 Sultana F, Wahab MA, Nahiduzzaman M, Mohiuddin M, Iqbal MZ, Shakil A, Mamun A-A, Khan MSR, Wong L, Asaduzzaman M (2023) Seaweed farming for food and nutritional security, climate change mitigation and adaptation, and women empowerment: A review. Aquaculture and Fisheries 8 (5):463-480. doi:https://doi.org/10.1016/j.aaf.2022.09.001 Suthar P, Gajaria TK, Reddy CRK (2019) Production of quality seaweed biomass through nutrient optimization for the sustainable land-based cultivation. Algal Research 42:101583. doi:https://doi.org/10.1016/j.algal.2019.101583 Titlyanov EA, Titlyanova TV (2010) Seaweed cultivation: Methods and problems. Russian Journal of Marine Biology 36 (4):227-242. doi:10.1134/S1063074010040012 Vasconcelos M, Marson GV, Turgeon S, Tamigneaux E, Beaulieu L (2022) Environmental conditions influence on the physicochemical properties of wild and cultivated Palmaria palmata in the Canadian Atlantic shore. Journal of Applied Phycology 34 (5):2565-2578 Wang Y, Feng Y, Liu X, Zhong M, Chen W, Wang F, Du H (2018) Response of Gracilaria lemaneiformis to nitrogen deprivation. Algal research 34:82-96 Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. Journal of plant physiology 144 (3):307-313 Werner A, Dring M (2011) Aquaculture explained no. 27-cultivating Palmaria palmata. Bord Iascaigh Mhara 76 Xu N, Xu K, Wang W, Xu Y, Ji D, Chen C, Xie C (2020) Nutrient enrichment improves growth and food quality of two strains of the economic seaweed Pyropia haitanensis. Frontiers in Marine Science 7:544582 Yong WTL, Thien VY, Misson M, Chin GJWL, Said Hussin SNI, Chong HLH, Yusof NA, Ma NL, Rodrigues KF (2024) Seaweed: A bioindustrial game-changer for the green revolution. Biomass and Bioenergy 183:107122. doi:https://doi.org/10.1016/j.biombioe.2024.107122 Young EB, Dring MJ, Savidge G, Birkett DA, Berges JA (2007) Seasonal variations in nitrate reductase activity and internal N pools in intertidal brown algae are correlated with ambient nitrate concentrations. Plant, cell & environment 30 (6):764-774 Zhao L-S, Li K, Wang Q-M, Song X-Y, Su H-N, Xie B-B, Zhang X-Y, Huang F, Chen X-L, Zhou B-C (2017) Nitrogen starvation impacts the photosynthetic performance of Porphyridium cruentum as revealed by chlorophyll a fluorescence. Scientific Reports 7 (1):8542 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 03 Oct, 2024 Reviews received at journal 22 Sep, 2024 Reviewers agreed at journal 09 Sep, 2024 Reviews received at journal 08 Sep, 2024 Reviewers agreed at journal 01 Sep, 2024 Reviewers agreed at journal 29 Aug, 2024 Reviewers agreed at journal 29 Aug, 2024 Reviewers invited by journal 29 Aug, 2024 Editor assigned by journal 29 Aug, 2024 Submission checks completed at journal 27 Aug, 2024 First submitted to journal 21 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4953297","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":357586143,"identity":"120f57c1-07d4-4378-8427-ba5fc43cf26e","order_by":0,"name":"Jae Woo Jung","email":"","orcid":"","institution":"Incheon National University","correspondingAuthor":false,"prefix":"","firstName":"Jae","middleName":"Woo","lastName":"Jung","suffix":""},{"id":357586144,"identity":"5d1c71e3-da1a-4078-ace2-3c3cce0d398d","order_by":1,"name":"Muriel Dittrich","email":"","orcid":"","institution":"University of Alaska Fairbanks","correspondingAuthor":false,"prefix":"","firstName":"Muriel","middleName":"","lastName":"Dittrich","suffix":""},{"id":357586145,"identity":"824f0d28-13a8-4e8a-8571-d972e5161db9","order_by":2,"name":"Jang K. Kim","email":"","orcid":"","institution":"Incheon National University","correspondingAuthor":false,"prefix":"","firstName":"Jang","middleName":"K.","lastName":"Kim","suffix":""},{"id":357586146,"identity":"b0f8b724-5ece-4ea8-8e0b-a55addeed195","order_by":3,"name":"Schery Umanzor","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYDADfgbmBgaGAyBmApFaJBsYSdVicIBYLfIzshM/F/yxyTc+frDx448zdgz87DkG+A2/kbtZegZPmuW2M4nN0jw3khkke94Q0CKRu0GaR+KwgdmBxAZphg/MQEMI2CI/I3fzbx6DwwbG/Q+bf/74UM9gT0gLw43cbdI8CYcNDCQS2yR4bhwG2kvIL2febrPmOZBmIHHjYZs1z5njPBJnnhXgd1h77ubbPH9sDPj7kw/f/HGsWo6/PXkDfoehAx7SlI+CUTAKRsEowAoAD+VJDasTYWkAAAAASUVORK5CYII=","orcid":"","institution":"University of Alaska Fairbanks","correspondingAuthor":true,"prefix":"","firstName":"Schery","middleName":"","lastName":"Umanzor","suffix":""}],"badges":[],"createdAt":"2024-08-21 17:38:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4953297/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4953297/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65354217,"identity":"00d4cf79-1008-4700-aef4-b5605bde3703","added_by":"auto","created_at":"2024-09-26 11:43:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":145856,"visible":true,"origin":"","legend":"\u003cp\u003eSpecific growth rate (SGR) of (A) \u003cem\u003eDevaleraea mollis \u003c/em\u003eand (B) \u003cem\u003ePalmaria hecatensis \u003c/em\u003eexposed to ambient seawater (AS), von Stosch enrichment medium (VSE), Guillard's f/2 medium (f/2), Jack's Special (JS) and JS with vitamin (JSV). Uppercase or lowercase letters represent significant differences among nutrient supplement treatments within the same period. Asterisk and double asterisks indicate significant differences between days 7 and 14 within nutrient supplement treatments at p \u0026lt; 0.05 and p \u0026lt; 0.01, respectively. Standard deviations are represented as a mean of n = 4.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4953297/v1/12ed5f7063cd4205dfa65a22.png"},{"id":65354426,"identity":"c31a5f5b-acad-49d3-9259-6b8501bbc249","added_by":"auto","created_at":"2024-09-26 11:51:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":354736,"visible":true,"origin":"","legend":"\u003cp\u003eTissue carbon (C), nitrogen (N) content and C:N ratio of \u003cem\u003eDevaleraea\u003c/em\u003e \u003cem\u003emollis\u003c/em\u003eand \u003cem\u003ePalmaria hecatensis \u003c/em\u003eexposed to ambient seawater (AS), von Stosch enrichment medium (VSE), Guillard's f/2 medium (f/2), Jack's Special (JS) and JS with vitamin (JSV). Uppercase or lowercase letters represent significant differences among nutrient supplement treatments within the same period. Asterisk and double asterisks indicate significant differences between days 0 and 14 within nutrient supplement treatments at p \u0026lt; 0.05 and p \u0026lt; 0.01, respectively. Standard deviations are represented as a mean of n = 4.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4953297/v1/823873966b69ec8b474b3b98.png"},{"id":65354215,"identity":"c6f64c36-410f-4169-b54c-eb2b35d6b156","added_by":"auto","created_at":"2024-09-26 11:43:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155662,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum quantum yield of PS Ⅱ (Fv/Fm) of (A) \u003cem\u003eDevaleraea mollis \u003c/em\u003eand (B) \u003cem\u003ePalmaria hecatensis \u003c/em\u003eexposed to ambient seawater (AS), von Stosch enrichment medium (VSE), Guillard's f/2 medium (f/2), Jack's Special (JS) and JS with vitamin (JSV). Uppercase or lowercase letters represent significant differences among nutrient supplement treatments within the same period. Asterisk and double asterisks indicate significant differences between days 0 and 14 within nutrient supplement treatments at p \u0026lt; 0.05 and p \u0026lt; 0.01, respectively. Standard deviations are represented as a mean of n = 4.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4953297/v1/2fd3162dfb1c1c71f52329a6.png"},{"id":65353298,"identity":"a7007821-83e2-4ead-952c-1b9609de94ba","added_by":"auto","created_at":"2024-09-26 11:35:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":266776,"visible":true,"origin":"","legend":"\u003cp\u003eChlorophyll a content of (A) \u003cem\u003eDevaleraea mollis \u003c/em\u003eand (B) \u003cem\u003ePalmaria hecatensis \u003c/em\u003eexposed to ambient seawater (AS), von Stosch enrichment medium (VSE), Guillard's f/2 medium (f/2), Jack's Special (JS) and JS with vitamin (JSV). Uppercase or lowercase letters represent significant differences among nutrient supplement treatments within the same period. Asterisk and double asterisks indicate significant differences between days 0 and 14 within nutrient supplement treatments at levels of p \u0026lt; 0.05 and p \u0026lt; 0.01, respectively. Standard deviations are represented as a mean of n = 4.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4953297/v1/6e1dd9a18333bd80f54d32f5.png"},{"id":65353302,"identity":"3670ec00-5df3-4f07-961e-410edf4a0b90","added_by":"auto","created_at":"2024-09-26 11:35:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":229951,"visible":true,"origin":"","legend":"\u003cp\u003eThe total protein content of (A) \u003cem\u003eDevaleraea mollis \u003c/em\u003eand (B) \u003cem\u003ePalmaria hecatensis \u003c/em\u003eexposed to ambient seawater (AS), von Stosch enrichment medium (VSE), Guillard's f/2 medium (f/2), Jack's Special (JS) and JS with vitamin (JSV). Uppercase or lowercase letters represent significant differences among nutrient supplement treatments within the same period. Asterisk and double asterisks indicate significant differences between days 0 and 14 within nutrient supplement treatments at p \u0026lt; 0.05 and p \u0026lt; 0.01, respectively. Standard deviations are represented as a mean of n = 4.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4953297/v1/730b89b46bab455d575c868c.png"},{"id":65353299,"identity":"3941ccef-e888-4a7a-a454-6e66d8329599","added_by":"auto","created_at":"2024-09-26 11:35:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":364172,"visible":true,"origin":"","legend":"\u003cp\u003eThe phycobiliprotein (phycocyanin and phycoerythrin)content of \u003cem\u003eDevaleraea mollis \u003c/em\u003eand \u003cem\u003ePalmaria hecatensis\u003c/em\u003e exposed to ambient seawater (AS), von Stosch enrichment medium (VSE), Guillard's f/2 medium (f/2), Jack's Special (JS) and JS with vitamin (JSV). Uppercase or lowercase letters represent significant differences among nutrient supplement treatments within the same period. Asterisk and double asterisks indicate significant differences between days 0 and 14 within nutrient supplement treatments at p \u0026lt; 0.05 and p \u0026lt; 0.01, respectively. Standard deviations are represented as a mean of n = 4.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4953297/v1/d0354e075ae028b38004242e.png"},{"id":65355219,"identity":"ca39d27c-cb67-459a-a31e-54cdeb2baee0","added_by":"auto","created_at":"2024-09-26 11:59:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2080124,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4953297/v1/fe54a46f-9369-44ab-a244-f7e3bee944de.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring Nutrient Supplements for Enhanced Growth and Quality of Devaleraea mollis and Palmaria hecatensis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e Seaweed aquaculture is one of the fastest-growing maritime industries worldwide (Kim et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sultana et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In 2022, 36.5\u0026nbsp;million tons of seaweed were produced with a value of 17\u0026nbsp;billion US\u003cspan\u003e$\u003c/span\u003e (FAO FishstatJ database). Most seaweed produced is directed to the food industry, including direct consumption, polysaccharide additives, and functional ingredients (Kim et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Naylor et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hydrocolloids extracted from seaweed stand as the second most valuable segment, while fertilizers and animal feed additives make up the remainder (Barbier et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Porse and Rudolph \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Shannon and Abu-Ghannam \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Naylor et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A global shift towards holistic health and sustainable food sources has recently increased demand for seaweed biomass, positioning it as a staple in functional foods and feeds rich in essential nutrients (Shannon and Abu-Ghannam \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yong et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Pacific Northwest of the United States is emerging as an optimal hub for the burgeoning seaweed farming industry (Considine et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). While the focus lies on kelp cultivation at sea, other types of seaweed farming are also explored. For example, the red seaweeds \u003cem\u003eDevaleraea mollis\u003c/em\u003e (formerly known as \u003cem\u003ePalmaria mollis\u003c/em\u003e) and \u003cem\u003ePalmaria hecatensis\u003c/em\u003e have gained interest as potential species for land-based cultivation given their high protein content (Demetropoulos and Langdon \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Saunders et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Gadberry et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eD. mollis\u003c/em\u003e has seen successful growth in outdoor land-based systems across Oregon, California, Hawaii, and more recently indoors in SE Alaska. Cultivation has mainly benefited the abalone industry along the Pacific coast (Evans and Langdon \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Demetropoulos and Langdon \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e; Langdon et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Evans et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). On the other hand, \u003cem\u003eP. hecatensis\u003c/em\u003e represents a novel aquaculture species for Alaska, with indoor land-based cultivation protocols recently developed (Dittrich et al. in prep).\u003c/p\u003e \u003cp\u003eStudies on \u003cem\u003ePalmaria palmata\u003c/em\u003e, a similar species from the Atlantic, highlight opportunities for researching downstream applications for \u003cem\u003eD. mollis\u003c/em\u003e and \u003cem\u003eP. hecatensis\u003c/em\u003e beyond abalone farming.\u003c/p\u003e \u003cp\u003e(Werner and Dring \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Corey et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rizzo et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eP. palmata\u003c/em\u003e is a top candidate for human consumption (Mouritsen et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Skriptsova and Kalita \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Skriptsova et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; St\u0026eacute;vant et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It boasts high protein levels ranging from 8\u0026ndash;35% of dry weight, contains various essential nutrients, polyunsaturated fatty acids like EPA, and antioxidants, and is reported to have anticancer properties, making it attractive for biomedical research (Morgan et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Mouritsen et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Albertos et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lopes et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Foseid et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Currently, the species is mainly grown on land, with cultivation in Ireland, Canada, Iceland, Norway, and the United States, among others, focusing on a consistent, high-quality supply for the increasing demand in the food and health sectors (Kim et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; St\u0026eacute;vant et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn contrast with farming at sea, one significant advantage of land-based cultivation is the ability to control and optimize seaweed growing conditions. Such controls can facilitate customizing biomass production to ensure high-quality standards and biosafety (Hafting et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hafting et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Barbier et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pereira et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, fine-tuning environmental factors such as light quality and quantity, temperature, salinity, pH, water turbulence, and nutrient sources plus their ideal concentrations may increase costs, restricting entry and large-scale production (Titlyanov and Titlyanova \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kim and Yarish \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Suthar et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ara\u0026uacute;jo et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ongoing research aims to develop cost-effective methods to maintain these optimal conditions without compromising biomass quality.\u003c/p\u003e \u003cp\u003eNutrient supplementation can become a significant operational cost at scale (Kim and Yarish \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For example, von Stosch enrichment medium (VSE), the recommended nutrient medium for indoor cultivation of red seaweeds (Ott \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Corey et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Redmond et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), can cost up to \u003cspan\u003e$\u003c/span\u003e20 per liter of solution becoming cost-prohibitive for commercial-scale production (Kim and Yarish \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Interestingly, despite containing essential nutrients such as nitrate, phosphate, iron, and vitamins, small-scale cultivation trials on \u003cem\u003eD. mollis\u003c/em\u003e and \u003cem\u003eP. hecatensis\u003c/em\u003e indicate that VSE may not be the most effective enriched medium for the cultivation of these seaweed species (Dittrich et al. in prep). Results were based on qualitative differences observed on thalli grown using VSE, f/2, and Jack's Special (JS) as nutrient sources. Comparable results have been reported for other red seaweed grown in tank conditions. For example, a study on \u003cem\u003eGracilaria tikvahiae\u003c/em\u003e compared the effectiveness of low-cost fertilizers versus VSE as a nutrient source. The findings revealed that JS, a commercially available fertilizer, produced growth rates and productivity comparable to VSE (Kim and Yarish \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Of relevance, the cost of JS was reported to be 98% lower than VSE, highlighting its economic advantage over reagent-grade nutrient sources.\u003c/p\u003e \u003cp\u003eNotably, nitrogen (N) plays a crucial role in seaweed development, with nitrate and ammonium being primary sources in nutrient supplements (Lobban and Harrison \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The effectiveness of nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) and ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) as nitrogen sources is context-dependent and species-specific (Lobban and Harrison \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Corey et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). For instance, studies conducted on \u003cem\u003eD. mollis\u003c/em\u003e co-cultured with abalone showed that nitrate, as a nitrogen source, promoted more growth than ammonium when assessed long-term (9 weeks). However, thalli exposed to ammonium showed more growth in the short term (2\u0026ndash;5 weeks) than those exposed to nitrate (Demetropoulos and Langdon \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004b\u003c/span\u003e). Therefore, in this study, we assessed whether different nutrient supplements with nitrogen incorporated as nitrate, ammonium, or both influence growth rates, photosynthesis efficiency, pigment, protein content, and nutrient content in tissue and medium of \u003cem\u003eD. mollis\u003c/em\u003e and \u003cem\u003eP. hecatensis\u003c/em\u003e. We hypothesized that (i) commercial fertilizers can replace the VSE medium for cultivating both species and (ii) different nitrogen sources will have different effects on growth and overall performance of both species. We seek to reduce operational and management costs associated with controlling culturing conditions of \u003cem\u003eD. mollis\u003c/em\u003e and \u003cem\u003eP. hecatensis\u003c/em\u003e without compromising biomass growth and quality in land-based tank cultivation systems.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample collection and pre-cultivation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eDevaleraea mollis\u003c/em\u003e and \u003cem\u003ePalmaria hecatensis\u003c/em\u003e were collected from Kodiak Island, Alaska. Both species were acclimated and cultured in a seawater flow-through tank in the Mariculture Lab at the Juneau College of Fisheries and Ocean Sciences. Salinity, temperature, and irradiance were maintained at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ppt, 8℃, and 100 photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e before the experimental period. Guillard's f/2 medium (f/2) was used as a nutrient supplement (Guillard \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1975\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental treatments\u003c/h2\u003e \u003cp\u003eThree different nutrient supplements, 1) von Stosch enrichment medium (VSE; Ott \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1965\u003c/span\u003e); 2) Guillard's f/2 medium (f/2); 3) commercial fertilizer, Jack's Special (25-5-15; JS), were used in the present study. Ambient seawater (AS) was used as a control without a nutrient supplement. Since VSE and f/2 contained vitamins, JS with vitamin (JSV) was also added to the experimental treatments. Thus, five experimental treatments (i.e., S, VSE, f/2, JS, and JSV), were used. Total nitrogen concentrations in f/2, JS, and JSV were adjusted to be the same as those in VSE (500 \u0026micro;M; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The experiment was conducted after seven consecutive days of nitrogen (N) starvation in ambient seawater without any nutrient supplementation. After that, 0.20 g of fresh weight of \u003cem\u003eDevaleraea mollis\u003c/em\u003e and \u003cem\u003ePalmaria hecatensis\u003c/em\u003e were transferred to independent 250 mL Erlenmeyer flasks filled with an assigned experimental treatment. Each experimental treatment had four replicates. Samples were cultivated in their assigned experimental treatment at 30 ppt, 8 ℃, 100 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e photosynthetically active radiation (PAR) and 12:12 h (L:D) photoperiod for two weeks. The culturing medium containing the targeted nutrient supplement was renewed every five days to avoid contaminant growth and potential unintended nutrient limitation.\u003c/p\u003e \u003cp\u003eThe specific growth rate (SGR) of each species was determined using the fresh weight of each sample as a metric. Fresh weight was measured every seven days within the 14 day experimental period. The short experimental period follows our interest in producing the most biomass in the shortest period possible before growth rates of parental thalli decrease (i.e., typically by day 21st after initiating cultures; Dittrich et al. in prep). It is also an adequate timeframe for measuring short-term physiological responses due to the experimental treatments.\u003c/p\u003e \u003cp\u003eThe specific growth rate of experimental thalli was calculated using the equation as follows (Krzemińska et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:SGR\\left(\\%\\:{day}^{-1}\\right)=\\frac{ln{Wt}_{2}-ln{Wt}_{1}}{{t}_{2}-{t}_{1}}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere Wt\u003csub\u003e2\u003c/sub\u003e and Wt\u003csub\u003e1\u003c/sub\u003e represent the weights of the thalli on days t\u003csub\u003e2\u003c/sub\u003e and t\u003csub\u003e1\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMicro and macroelement concentration in different nutrient supplement\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNutrient (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVSE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ef/2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJSV\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNitrogen\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e(100% NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e(100% NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e(57% NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e and 43% NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e(57% NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e and 43% NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePhosphorus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIron\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eManganese\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEDTA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVitamins\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVitamin B\u003csub\u003e1\u003c/sub\u003e and B\u003csub\u003e12\u003c/sub\u003e, Biotin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVitamin B\u003csub\u003e1\u003c/sub\u003e and B\u003csub\u003e12\u003c/sub\u003e, Biotin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVitamin B\u003csub\u003e1\u003c/sub\u003e and B\u003csub\u003e12\u003c/sub\u003e, Biotin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePotassium\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e107\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBoron\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCopper\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMolybdenum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eZinc\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMagnesium\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Nutrient analysis\u003c/h2\u003e \u003cp\u003eApproximately 50 mg of fresh thalli was collected on day 0 (start) and day 14 (end) of the experiment and dried in a dry oven at 60 ℃ until constant weight. Dried samples were ground to powder using an MM400 Ball Mill (Retsch, Germany). The tissue carbon and nitrogen content of each sample was analyzed using a CHN analyzer (Thermo Fisher, USA). Moreover, water samples from each nutrient supplement stock and experimental treatment were collected and filtered through 0.45 \u0026micro;m syringed filters (33mm diameter, Chromdisc, Korea) when the media was renewed. The concentration of nitrate (nitrite), ammonium, and phosphorus was measured using a Continuous Segmented Flow Analyzer (QuAAtro 39, SEAL Analytical).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Photosynthesis efficiency and chlorophyll-a\u003c/h2\u003e \u003cp\u003ePhotosynthesis efficiency and chlorophyll-a content were measured on day 0 and day 14 of the experiment. The minimum fluorescence (Fo) and maximum fluorescence (Fm) were measured by pulse amplitude modulated fluorometry (Junior-PAM; Walz, Germany). The maximum quantum yield of PS Ⅱ (Fv/Fm) was calculated as Fv/Fm = (Fm-Fo)/Fm. Chlorophyll-a content was determined using an extract prepared with approximately 20 mg of fresh thalli placed in 2 mL of ice-cold methanol (95%) and kept at 4 ℃ for 24h at dark. Light absorbance of the extract was measured at 666 and 653 nm and calculated as mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Total protein and phycobiliprotein\u003c/h2\u003e \u003cp\u003eTotal protein content was measured according to the Bradford protein assay at day 0 and day 14 of the experiment (Bradford \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). Approximately 50 mg of fresh thalli were homogenized with 1 mL of potassium phosphate buffer (50mM, pH7) containing 0.25% Triton X-100 and 1% polyvinylpyrrolidone in cold. The homogenates were centrifuged at 12000 g for 10 minutes at 4 ℃. Bradford's reagent (1 mL) was then added to 100 \u0026micro;L of supernatant and incubated at room temperature for 5 minutes. Absorbance was measured at 595 nm within 1 hour. Total protein contents were calculated as mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh weight. Bovine serum albumin was used as a standard.\u003c/p\u003e \u003cp\u003ePhycobiliproteins (phycoerythrin and phycocyanin) were also measured on day 0 and day 14 of the experiment (Beer and Eshel \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Approximately 50 mg of fresh thalli was ground up with 2.5 mL sodium phosphate buffer (0.1M, pH6.5) and kept at 4 ℃ for 24 hours in darkness. The mixture was centrifuged at 19000g for 20 minutes. The supernatant was measured at 455, 564, 592, 618, and 645 nm and expressed as mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eOne-way ANOVA and Tukey's HSD test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were conducted to check for statistical differences among nutrient supplements at day 0 and day 14 of the experiment. A t-test was used to detect significant differences between day 0 and day 14 of the experiment within samples exposed to the same nutrient supplement. All data were checked for normality using the Kolmogorov-Smirnov test and homogeneity of variance using Levene's test. Data were analyzed using the statistical software SPSS 25.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe specific growth rate (SGR) of \u003cem\u003eDevaleraea mollis\u003c/em\u003e on day 7 and day 14 showed significant differences due to the nutrient supplements (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and p\u0026thinsp;=\u0026thinsp;0.040, respectively). At day 7, the SGR of \u003cem\u003eD. mollis\u003c/em\u003e exposed to von Stosch enrichment medium (VSE) and Guillard's f/2 medium (f/2) were significantly higher than those exposed to ambient seawater (AS), Jack's Special (JS), and JS with vitamin (JSV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At day 14, differences were neglectable, with the only significant difference detected between SGRs of samples exposed to f/2 versus AS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, results show a significant decrease in SGR of samples exposed to AS and VSE between day 7 and day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; p\u0026thinsp;=\u0026thinsp;0.040 and p\u0026thinsp;=\u0026thinsp;0.034, respectively). On the other hand, the SGR of \u003cem\u003ePalmaria hecatensis\u003c/em\u003e was not affected by the experimental treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Through the experiment, \u003cem\u003eD. mollis\u003c/em\u003e showed a faster growth rate than \u003cem\u003eP. hecatensis\u003c/em\u003e, with SGR of the former more than 300% of the latter.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough results varied, the experimental treatments also influenced tissue nitrogen (N) content and carbon:nitrogen (C:N) ratio for both species. The tissue nitrogen (N) content of \u003cem\u003eD. mollis\u003c/em\u003e exposed to f/2, JS, and JSV was significantly higher than those growing in AS and VSE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. The tissue C:N ratio followed the opposite trend, with thalli exposed to AS and VSE showing significantly higher than those in f/2, JS, and JSV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). On the other hand, tissue nitrogen content of \u003cem\u003eP. hecatensis\u003c/em\u003e showed significant differences only between JSV and AS, with JSV showing higher values (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE; p\u0026thinsp;=\u0026thinsp;0.019). The tissue C:N ratio of \u003cem\u003eP. hecatensis\u003c/em\u003e exposed to JS and JSV was significantly lower than thalli exposed to AS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF; p\u0026thinsp;=\u0026thinsp;0.014 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). Experimental treatments did not significantly influence tissue C content for either species (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBy days 0 and 14, mean dissolved inorganic nitrogen (DIN) and phosphate (DIP) uptake rates (\u0026micro;M day 7⁻\u0026sup1;) of \u003cem\u003eD. mollis\u003c/em\u003e exposed to VSE were significantly higher than those exposed to f/2, JS, and JSV (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Conversely, the mean DIN uptake rates of \u003cem\u003eP. hecatensis\u003c/em\u003e exposed to VSE were significantly lower than those of thalli exposed to f/2, JS, and JSV at both sampling periods (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For \u003cem\u003eD. mollis\u003c/em\u003e, the mean DIN:DIP uptake ratio was 16:1 (VSE), 25:1 (f/2), and 23:1 (JS and JSV) by day 7. These ratios showed slight variation by day 14 of the experiment, with values of 15:1 (VSE), 26:1 (f/2), 20:1, and 21:1 (JSV) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For \u003cem\u003eP. hecatensis\u003c/em\u003e, the mean DIN:DIP uptake ratio was 12:1 (VSE), 15:1 (f/2), 14:1 (JS and JSV) by day 7. These ratios also showed slight variation by day 14 of the experiment, with values of 11:1 (VSE), 15:1 (f/2) 12:1 and 13:1 (JSV) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDissolved inorganic nitrogen (DIN) and phosphate (DIP) uptake and their ratio of \u003cem\u003eDevaleraea mollis\u003c/em\u003e exposed to ambient seawater (AS), von Stosch enrichment medium (VSE), Guillard's f/2 medium (f/2), Jack's Special (JS), and JS with vitamin (JSV). Letters represent significant differences among nutrient supplement treatments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eNutrient uptake (\u0026micro;M day 7\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVSE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ef/2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eJS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eJSV\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDIN\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e490.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e432.72\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e459.62\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e461.47\u0026thinsp;\u0026plusmn;\u0026thinsp;3.17 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e490.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e432.72\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e260.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e259.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmmonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e199.36\u0026thinsp;\u0026plusmn;\u0026thinsp;3.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e205.21\u0026thinsp;\u0026plusmn;\u0026thinsp;6.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDIP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.02\u0026thinsp;\u0026plusmn;\u0026thinsp;4.66 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.13\u0026thinsp;\u0026plusmn;\u0026thinsp;3.13 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN : P ratio\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e23:1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDIN\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e490.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e433.24\u0026thinsp;\u0026plusmn;\u0026thinsp;6.55 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e464.39\u0026thinsp;\u0026plusmn;\u0026thinsp;8.42 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e461.95\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e490.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e433.24\u0026thinsp;\u0026plusmn;\u0026thinsp;6.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e260.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e258.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmmonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e200.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e203.04\u0026thinsp;\u0026plusmn;\u0026thinsp;4.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDIP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23.05\u0026thinsp;\u0026plusmn;\u0026thinsp;3.58 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.58 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN : P ratio\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21:1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDissolved inorganic nitrogen (DIN) and phosphate (DIP) uptake and ratio of \u003cem\u003ePalmaria hecatensis\u003c/em\u003e at ambient seawater (AS), von Stosch enrichment medium (VSE), Guillard's f/2 medium (f/2), Jack's Special (JS) and JS with vitamin (JSV). Different letters represent the significant difference among nutrient supplements.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eNutrient uptake (\u0026micro;M day 7\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVSE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ef/2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eJS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eJSV\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDIN\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.88\u0026thinsp;\u0026plusmn;\u0026thinsp;7.92\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e66.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.71\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e62.54\u0026thinsp;\u0026plusmn;\u0026thinsp;5.74\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e58.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.88\u0026thinsp;\u0026plusmn;\u0026thinsp;7.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e66.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e48.64\u0026thinsp;\u0026plusmn;\u0026thinsp;6.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e45.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmmonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDIP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN : P ratio\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14:1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDIN\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.23\u0026thinsp;\u0026plusmn;\u0026thinsp;8.86 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e69.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e57.15\u0026thinsp;\u0026plusmn;\u0026thinsp;8.25 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60.98\u0026thinsp;\u0026plusmn;\u0026thinsp;4.71 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.23\u0026thinsp;\u0026plusmn;\u0026thinsp;8.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e69.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e44.20\u0026thinsp;\u0026plusmn;\u0026thinsp;8.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e47.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmmonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDIP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eN : P ratio\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13:1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe maximum quantum yield of PS Ⅱ (Fv/Fm) of \u003cem\u003eD. mollis\u003c/em\u003e and \u003cem\u003eP. hecatensis\u003c/em\u003e was not affected by the experimental nutrient supplements (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConversely, results show a significant difference in chlorophyll-a content due to the experimental treatments in \u003cem\u003eD. mollis\u003c/em\u003e and \u003cem\u003eP. hecatensis\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The chlorophyll-a content in \u003cem\u003eD. mollis\u003c/em\u003e exposed to f/2, JS, and JSV were significantly higher than those of thalli exposed to AS at day 14 of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, chlorophyll-a content of \u003cem\u003eD. mollis\u003c/em\u003e exposed to f/2, JS, and JSV was significantly higher on day 14 of the experiment than on day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the other hand, chlorophyll-a content of \u003cem\u003eP. hecatensis\u003c/em\u003e exposed to VSE, f/2, JS, and JSV were also significantly higher than those thalli exposed to AS at day 14 of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). On day 14 of the experiment, chlorophyll-a content of \u003cem\u003eP. hecatensis\u003c/em\u003e exposed to AS was significantly lower than that at day 0 of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; p\u0026thinsp;=\u0026thinsp;0.005). No significant difference in chlorophyll-a between day 0 and day 14 of the experiment was detected for any other treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, nutrient supplement treatments significantly influenced the total protein content of D. mollis and P. hecatensis (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The total protein content of \u003cem\u003eD. mollis\u003c/em\u003e exposed to f/2, JS, and JSV were significantly higher than those at AS and VSE by day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Results show a significant decrease in the total protein content of \u003cem\u003eD. mollis\u003c/em\u003e exposed AS and VSE between day 0 and day 14 of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, samples exposed to JS and JSV showed a significant increase on day 14 compared to day 0 of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eP. hecatensis\u003c/em\u003e also showed significantly different responses, with samples exposed to VSE, f/2, JS, and JSV having a higher protein content than those exposed to AS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All treatments, except for AS, showed significantly higher protein content at day 14 versus day 0 of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, with protein content, the phycobiliprotein content (phycocyanin and phycoerythrin) of \u003cem\u003eD. mollis\u003c/em\u003e and \u003cem\u003eP. hecatensis\u003c/em\u003e followed different trends. Phycobiliprotein content in \u003cem\u003eD. mollis\u003c/em\u003e was significantly affected by the experimental treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while the content in \u003cem\u003eP. hecatensis\u003c/em\u003e was not (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Specifically, phycocyanin and phycoerythrin in \u003cem\u003eD. mollis\u003c/em\u003e exposed to f/2, JS, and JSV were significantly higher than those exposed to AS and VSE by day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). On day 14 of the experiment, phycocyanin and phycoerythrin of \u003cem\u003eD. mollis\u003c/em\u003e exposed to AS were significantly lower, whereas thalli exposed to f/2, JS, and JSV were significantly higher by day 14 compared to day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOn-land tank seaweed cultivation offers numerous benefits, including improved control over biomass quality. This cultivation technique has demonstrated the highest yields per square meter of water surface compared to other methods (Titlyanov and Titlyanova \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Hwang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shin et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, we investigated whether the cost of tank cultivation could be reduced without compromising the growth and performance of the red seaweeds \u003cem\u003eDevalaraea mollis\u003c/em\u003e and \u003cem\u003ePalmaria hecatensis\u003c/em\u003e by using commercial-grade nutrient supplementation instead of the recommended reagent-grade nutrient source.\u003c/p\u003e \u003cp\u003eOur results showed that the commercial fertilizer Jack's Special (JS) effectively replaced the von Stosch enrichment medium (VSE), favoring growth and protein content of both species. As expected, there were differences in crop performance based on nutrient source and species over time. These findings align with previous cultivation trials where the reagent-grade product, VSE, showed limited benefits for the growth performance of \u003cem\u003eD. mollis\u003c/em\u003e and \u003cem\u003eP. hecatensis\u003c/em\u003e (Dittrich et al. in prep).\u003c/p\u003e \u003cp\u003eIn this study, we focused on nitrogen, an essential element for seaweed growth and often a limiting factor (Kim et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hurd et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Roleda and Hurd \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bao et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our results revealed that the Specific Growth Rate (SGR) of \u003cem\u003eD. mollis\u003c/em\u003e exposed to ambient seawater (AS) and VSE significantly decreased from day 7 to day 14. The carbon to nitrogen ratio (C:N), a key indicator of nitrogen status (Kim et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hurd et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), showed nitrogen limitation at day 14 of the experiment, with mean tissue C:N of 16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77 and 15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40 for thalli growing with AS and VSE, respectively. In contrast, the C:N of samples exposed to Guillard's f/2 medium (f/2), JS, and JS with vitamins (JSV) indicated nitrogen accumulation (9.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06, 8.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07, and 7.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56, respectively). This reduction in growth linked to nitrogen limitation aligns with studies conducted on \u003cem\u003ePyropia yezoensis\u003c/em\u003e, \u003cem\u003eGracilariopsis lemaneiformis\u003c/em\u003e, \u003cem\u003eGracilaria tikvahiae\u003c/em\u003e and \u003cem\u003eUlva pertusa\u003c/em\u003e (Liu and Dong \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kim and Yarish \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe SGR of \u003cem\u003eP. hecatensis\u003c/em\u003e was not affected by the type of nutrient supplementation. However, similar to \u003cem\u003eD. mollis\u003c/em\u003e, the mean tissue C:N of thalli exposed to JS and JSV indicated nitrogen accumulation (9.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 and 8.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19, respectively), which was significantly lower than in \u003cem\u003eP. hecatensis\u003c/em\u003e grown in AS. Under limiting conditions, seaweed obtain nitrogen through the catabolism of nitrogen-based compounds, leading to reduced concentrations of these compounds, such as chlorophyll-a, phycobiliproteins, and proteins (Kim et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Roleda and Hurd \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The catabolism of nitrogen-based compounds might maintain or increase growth in the short term, but ultimately, it might inhibit growth in the long term if the limitation persists.\u003c/p\u003e \u003cp\u003eOverall, our results indicate that JS and modified JS with vitamins can replace VSE and f/2 for growing \u003cem\u003eD. mollis\u003c/em\u003e and \u003cem\u003eP. hecatensis\u003c/em\u003e, with JS and JSV showing the highest protein content in \u003cem\u003eD. mollis\u003c/em\u003e. VSE and f/2 contain only nitrate as a nitrogen source, while JS and JSV contain both nitrate and ammonium (Kim and Yarish \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Ammonium contributed to higher protein content due to its direct incorporation into amino acids (Lobban and Harrison \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). On the other hand, although the targeted seaweed could have incorporated nitrate, the process was not conducive to promoting the most growth, as it required reduction for assimilation (Hurd et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Pritchard et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). First, nitrate reductase (NR) converts nitrate to nitrite (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e), and nitrite reductase (NiR) converts nitrite to ammonium. These processes require energy in the form of NADPH or ferredoxin, making the assimilation of nitrate more energetically costly compared to ammonium (Jin et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Cohen and Fong \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Additionally, although temperature was not an experimental factor in this study, it could have affected the enzymatic activity and active transport of nitrate, but not passive diffusion of ammonium (Nishihara et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Roleda and Hurd \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this case, ammonium transporters facilitate the rapid influx of ammonium ions into the cells, making it easier for the seaweed to utilize ammonium when available.\u003c/p\u003e \u003cp\u003eOur results show that the phycobiliprotein content of \u003cem\u003eD. mollis\u003c/em\u003e in JS and JSV was significantly higher than in AS. This finding aligns with the notion that seaweed can store excess nitrogen through pigments and other compounds, supporting future growth under nitrogen depletion (McGlathery et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Naldi and Wheeler \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). For instance, \u003cem\u003eGrateloupia turuturu\u003c/em\u003e stores extra ammonium in phycobiliprotein (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), while \u003cem\u003ePalmaria palmata\u003c/em\u003e can synthesize and accumulate phycoerythrin under nutrient-replete conditions (Furuta et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Schmedes and Nielsen \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Vasconcelos et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Here, \u003cem\u003eD. mollis\u003c/em\u003e showed protein profiles akin to those of \u003cem\u003ePalmaria palmata\u003c/em\u003e. Although the protein content of \u003cem\u003eP. hecatensis\u003c/em\u003e exposed to VSE, f/2, JS, and JSV was significantly higher than in AS, there were no differences in phycobiliprotein content among nutrient supplements. It has been documented that nitrogen storage pools can vary between species (Naldi and Wheeler \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Young et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mendez and Kwon \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which seems to be the case here. \u003cem\u003eP. hecatensis\u003c/em\u003e may likely require tailored nutrient management strategies to optimize their growth and protein content, highlighting the need for species-specific cultivation practices in on-land tank systems.\u003c/p\u003e \u003cp\u003eFinally, our results emphasize the need to consider the unique nutritional and storage requirements of targeted seaweed species to enhance productivity and quality. Further research is necessary to understand the specific nitrogen storage mechanisms and protein compositions of \u003cem\u003eP. hecatensis\u003c/em\u003e, which can lead to more effective fertilizers and growth media. These insights can inform species selection based on their growth characteristics and nutrient requirements for commercial seaweed production, potentially improving yield and reducing costs. Additionally, understanding species-specific responses to nutrient supplementation can inform ecological studies in the context of changing environmental conditions. Furthermore, the knowledge of distinct nitrogen storage pools and protein compositions can be leveraged in biotechnological applications, such as producing bioactive compounds, pigments, and other valuable products from red seaweed. Lastly, trace metals such as zinc and copper, essential for photosynthesis, growth, and cellular metabolism, also play a crucial role in seaweed cultivation (Howarth and Cole \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Demetropoulos and Langdon \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004b\u003c/span\u003e). Further studies on the effect of different trace metal concentrations among nutrient supplements would add to the increasing literature regarding the cultivation of red seaweeds.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, \u003cem\u003eD. mollis\u003c/em\u003e showed decreased growth when cultured with VSE due to nitrogen limitation, indicated by lower total protein content and higher tissue C:N ratio. The highest protein content was observed with the commercial fertilizer Jack's Special (25-5-15), likely due to the presence of ammonium. \u003cem\u003eP. hecatensis\u003c/em\u003e demonstrated similar growth, pigment, and protein content across all nutrient supplements, indicating that Jack's Special can effectively replace VSE for both species, reducing operational and management costs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical Approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available upon request to the authors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJW J conceptualized, investigated, conducted formal analysis, wrote the original draft, and reviewed and edited the manuscript. MD conceptualized, reviewed, and edited the manuscript. JK K and SU conceptualized, conducted formal analysis, reviewed and edited the manuscript, acquired funding, and supervised.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis study was funded by the Ministry of Education (NRF-2017R1A6A1A06015181), the Ministry of Science and ICT through the National Research Foundation of Korea (NRF-2021K1A3A1A05086015), the Ministry of Oceans and Fisheries of Korea (Project No. 20190518) and the State of Alaska (Project No. G00015020).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlbertos I, Martin-Diana A, Bur\u0026oacute;n M, Rico D (2019) Development of functional bio-based seaweed (Himanthalia elongata and Palmaria palmata) edible films for extending the shelflife of fresh fish burgers. Food Packaging and Shelf Life 22:100382\u003c/li\u003e\n\u003cli\u003eAndrade LR, Farina M, Amado Filho GM (2004) Effects of copper on Enteromorpha flexuosa (Chlorophyta) in vitro. Ecotoxicology and environmental safety 58 (1):117-125\u003c/li\u003e\n\u003cli\u003eAra\u0026uacute;jo R, V\u0026aacute;zquez Calder\u0026oacute;n F, S\u0026aacute;nchez L\u0026oacute;pez J, Azevedo IC, Bruhn A, Fluch S, Garcia Tasende M, Ghaderiardakani F, Ilmj\u0026auml;rv T, Laurans M (2021) Current status of the algae production industry in Europe: an emerging sector of the blue bioeconomy. Frontiers in Marine Science 7:626389\u003c/li\u003e\n\u003cli\u003eBao M, Xing Q, Park J-S, He P, Zhang J, Yarish C, Kim JK (2023) Temperature and high nutrients enhance hypo-salinity tolerance of the bloom forming green alga, Ulva prolifera. Harmful Algae 123:102402\u003c/li\u003e\n\u003cli\u003eBarbier M, Charrier B, Araujo R, Holdt S, Jacquemin B, Rebours C (2019) Phycomorph European guidelines for a sustainable aquaculture of seaweeds. COST action FA1406 doi org/1021411/2c3w-yc73\u003c/li\u003e\n\u003cli\u003eBarbier M, Charrier B, Araujo R, Holdt S, Jacquemin B, Rebours C, Abreu H, Bruhn A, De Clerck O, Funderund J (2018) PEGASUS: Phycomorph european guidelines for sustainable aquaculture of seaweeds. Centro Oceanogr\u0026aacute;fico de Santander\u003c/li\u003e\n\u003cli\u003eBeer S, Eshel A (1985) Determining phycoerythrin and phycocyanin concentrations in aqueous crude extracts of red algae. Aust J Mar Freshw Res 36 (6):785-792\u003c/li\u003e\n\u003cli\u003eBothe H (2011) Plants in heavy metal soils. Detoxification of heavy metals:35-57\u003c/li\u003e\n\u003cli\u003eBradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry 72 (1-2):248-254\u003c/li\u003e\n\u003cli\u003eChen Y, Lan L, Zhang J, Wang Q, Liu Y, Li H, Gong Q, Gao X (2023) Physiological Impacts of Nitrogen Starvation and Subsequent Recovery on the Red Seaweed Grateloupia turuturu (Halymeniaceae, Rhodophyta). Sustainability 15 (9):7032\u003c/li\u003e\n\u003cli\u003eCohen RA, Fong P (2004) Nitrogen uptake and assimilation in Enteromorpha intestinalis (L.) Link (Chlorophyta): using 15N to determine preference during simultaneous pulses of nitrate and ammonium. Journal of experimental marine biology and ecology 309 (1):67-77\u003c/li\u003e\n\u003cli\u003eColeman JE (1998) Zinc enzymes. Current opinion in chemical biology 2 (2):222-234\u003c/li\u003e\n\u003cli\u003eConsidine ME, Kirchner G, Carter J, Waters T (2023) Situation analysis for Oregon\u0026apos;s emergent seaweed aquaculture industry.\u003c/li\u003e\n\u003cli\u003eCorey P, Kim J, Garbary D, Prithiviraj B, Duston J (2012) Bioremediation potential of Chondrus crispus (Basin Head) and Palmaria palmata: effect of temperature and high nitrate on nutrient removal. Journal of Applied Phycology 24:441-448\u003c/li\u003e\n\u003cli\u003eCorey P, Kim J, Duston J, Garbary D (2014) Growth and nutrient uptake by Palmaria palmata (Palmariales, Rhodophyta) integrated with Atlantic halibut recirculating aquaculture. Algae 29:35-45\u003c/li\u003e\n\u003cli\u003eCorey P, Kim JK, Duston J, Garbary DJ, Prithiviraj B (2013) Bioremediation potential of Palmaria palmata and Chondrus crispus (Basin Head): effect of nitrate and ammonium ratio as nitrogen source on nutrient removal. Journal of applied phycology 25:1349-1358\u003c/li\u003e\n\u003cli\u003eDemetropoulos CL, Langdon CJ (2004a) Effects of nutrient enrichment and biochemical composition of diets of Palmaria mollis on growth and condition of Japanese abalone, Haliotis discus hannai and red abalone, Haliotis rufescens. Journal of experimental marine biology and ecology 308 (2):185-206\u003c/li\u003e\n\u003cli\u003eDemetropoulos CL, Langdon CJ (2004b) Enhanced production of Pacific dulse (Palmaria mollis) for co-culture with abalone in a land-based system: nitrogen, phosphorus, and trace metal nutrition. Aquaculture 235 (1-4):433-455\u003c/li\u003e\n\u003cli\u003eEvans F, Langdon CJ (2000) Co-culture of dulse Palmaria mollis and red abalone Haliotis rufescens under limited flow conditions. Aquaculture 185 (1-2):137-158\u003c/li\u003e\n\u003cli\u003eEvans S, Rorrer GL, Langdon CJ (2021) Cultivation of the macrophytic red alga Palmaria mollis (Pacific dulse) on vertical arrays of mesh panels in aerated tanks. Journal of Applied Phycology 33 (6):3915-3926. doi:10.1007/s10811-021-02582-1\u003c/li\u003e\n\u003cli\u003eFoseid L, Natvik I, Devle H, Ekeberg D (2020) Identification of fatty acids in fractionated lipid extracts from Palmaria palmata, Alaria esculenta and Saccharina latissima by off-line SPE GC-MS. Journal of Applied Phycology 32 (6):4251-4262\u003c/li\u003e\n\u003cli\u003eFredriksen S, Rueness J (1989) Culture studies of Gelidium latifolium (Grev.) Born et Thur.(Rhodophyta) from Norway. Growth and nitrogen storage in response to varying photon flux density, temperature and nitrogen availability.\u003c/li\u003e\n\u003cli\u003eFuruta T, Miyabe Y, Yasui H, Kinoshita Y, Kishimura H (2016) Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata. Marine Drugs 14 (2):32\u003c/li\u003e\n\u003cli\u003eGadberry BA, Colt J, Maynard D, Boratyn DC, Webb K, Johnson RB, Saunders GW, Boyer RH, Gadberry BA, Colt J (2018) Intensive land-based production of red and green macroalgae for human consumption in the Pacific Northwest: an evaluation of seasonal growth, yield, nutritional composition, and contaminant levels. Algae 33 (1):109-125\u003c/li\u003e\n\u003cli\u003eGuillard RR Culture of phytoplankton for feeding marine invertebrates. In: Culture of marine invertebrate animals: proceedings\u0026mdash;1st conference on culture of marine invertebrate animals greenport, 1975. Springer, pp 29-60\u003c/li\u003e\n\u003cli\u003eHafting JT, Craigie JS, Stengel DB, Loureiro RR, Buschmann AH, Yarish C, Edwards MD, Critchley AT (2015) Prospects and challenges for industrial production of seaweed bioactives. Journal of Phycology 51 (5):821-837\u003c/li\u003e\n\u003cli\u003eHafting JT, Critchley AT, Cornish ML, Hubley SA, Archibald AF (2012) On-land cultivation of functional seaweed products for human usage. Journal of Applied Phycology 24:385-392\u003c/li\u003e\n\u003cli\u003eHecky R, Kilham P (1988) Nutrient limitation of phytoplankton in freshwater and marine environments: a review of recent evidence on the effects of enrichment 1. Limnology and oceanography 33 (4part2):796-822\u003c/li\u003e\n\u003cli\u003eHowarth RW, Cole JJ (1985) Molybdenum availability, nitrogen limitation, and phytoplankton growth in natural waters. Science 229 (4714):653-655\u003c/li\u003e\n\u003cli\u003eHurd CL, Berges JA, Osborne J, Harrison PJ (1995) An in Vitro Nitrate Reductase Assay for Marine Macroalgae: Optimization and Characterization of the Enzyme for Fucus Gardneri (PHAEOPHYTA) 1. Journal of Phycology 31 (5):835-843\u003c/li\u003e\n\u003cli\u003eHurd CL, Harrison PJ, Bischof K, Lobban CS (2014) Seaweed ecology and physiology. Cambridge University Press\u003c/li\u003e\n\u003cli\u003eHwang EK, Choi HG, Kim JK (2020) Seaweed resources of Korea. Botanica Marina 63 (4):395-405\u003c/li\u003e\n\u003cli\u003eJin T, Huppe HC, Turpin DH (1998) In vitro reconstitution of electron transport from glucose-6-phosphate and NADPH to nitrite. Plant physiology 117 (1):303-309\u003c/li\u003e\n\u003cli\u003eKim J, Stekoll M, Yarish C (2019) Opportunities, challenges and future directions of open-water seaweed aquaculture in the United States. Phycologia 58 (5):446-461\u003c/li\u003e\n\u003cli\u003eKim JK, Duston J, Corey P, Garbary DJ (2013) Marine finfish effluent bioremediation: effects of stocking density and temperature on nitrogen removal capacity of Chondrus crispus and Palmaria palmata (Rhodophyta). Aquaculture 414:210-216\u003c/li\u003e\n\u003cli\u003eKim JK, Kraemer GP, Neefus CD, Chung IK, Yarish C (2007) Effects of temperature and ammonium on growth, pigment production and nitrogen uptake by four species of Porphyra (Bangiales, Rhodophyta) native to the New England coast. Journal of Applied Phycology 19:431-440\u003c/li\u003e\n\u003cli\u003eKim JK, Kraemer GP, Yarish C (2008) Physiological activity of Porphyra in relation to eulittoral zonation. Journal of Experimental Marine Biology and Ecology 365 (2):75-85\u003c/li\u003e\n\u003cli\u003eKim JK, Kraemer GP, Yarish C (2012) Metabolic plasticity of nitrogen assimilation by Porphyra umbilicalis (Linnaeus) K\u0026uuml;tzing. Journal of Ocean University of China 11 (4):517-526. doi:10.1007/s11802-012-2116-2\u003c/li\u003e\n\u003cli\u003eKim JK, Yarish C (2014) Development of a sustainable land-based Gracilaria cultivation system. Algae 29 (3):217-225\u003c/li\u003e\n\u003cli\u003eKim JK, Yarish C, Hwang EK, Park M, Kim Y, Kim JK, Yarish C, Hwang EK, Park M, Kim Y (2017) Seaweed aquaculture: cultivation technologies, challenges and its ecosystem services. Algae 32 (1):1-13\u003c/li\u003e\n\u003cli\u003eKrzemińska I, Pawlik-Skowrońska B, Trzcińska M, Tys J (2014) Influence of photoperiods on the growth rate and biomass productivity of green microalgae. Bioprocess and biosystems engineering 37:735-741\u003c/li\u003e\n\u003cli\u003eLangdon C, Evans F, Demetropoulos C (2004) An environmentally-sustainable, integrated, co-culture system for dulse and abalone production. Aquacultural engineering 32 (1):43-56\u003c/li\u003e\n\u003cli\u003eLi C, Ariga I, Mikami K (2019) Difference in nitrogen starvation-inducible expression patterns among phylogenetically diverse ammonium transporter genes in the red seaweed Pyropia yezoensis. American Journal of Plant Sciences 10 (8):1325-1349\u003c/li\u003e\n\u003cli\u003eLi YX, Zhou S, Zhao FJ, Liu Y, Fan PP, Wang GC (2010) Physiological responses of Porphyra haitanesis to different copper and zinc concentrations. Brazilian Journal of Oceanography 58:261-267\u003c/li\u003e\n\u003cli\u003eLiu J-W, Dong S-L (2001) Comparative studies on utilizing nitrogen capacity between two macroalgae Gracilaria tenuistipitata var. liui (Rhodophyta) and Ulva pertusa (Chlorophyta) I. Nitrogen storage under nitrogen enrichment and starvation. Journal of Environmental Sciences 13 (3):318-322\u003c/li\u003e\n\u003cli\u003eLiu X, Wen J, Chen W, Du H (2019a) Physiological effects of nitrogen deficiency and recovery on the macroalga Gracilariopsis lemaneiformis (Rhodophyta). Journal of Phycology 55 (4):830-839\u003c/li\u003e\n\u003cli\u003eLiu X, Wen J, Zheng C, Jia H, Chen W, Du H (2019b) The impact of nitrogen deficiency and subsequent recovery on the photosynthetic performance of the red macroalga Gracilariopsis lemaneiformis. Journal of Applied Phycology 31:2699-2707\u003c/li\u003e\n\u003cli\u003eLobban CS, Harrison PJ (1994) Seaweed ecology and physiology. Cambridge University Press,\u003c/li\u003e\n\u003cli\u003eLopes D, Melo T, Meneses J, Abreu MH, Pereira R, Domingues P, Lilleb\u0026oslash; AI, Calado R, Domingues MR (2019) A new look for the red macroalga Palmaria palmata: A seafood with polar lipids rich in EPA and with antioxidant properties. Marine drugs 17 (9):533\u003c/li\u003e\n\u003cli\u003eMamun M, Kwon S, Kim J-E, An K-G (2020) Evaluation of algal chlorophyll and nutrient relations and the N:P ratios along with trophic status and light regime in 60 Korea reservoirs. Science of The Total Environment 741:140451. doi:https://doi.org/10.1016/j.scitotenv.2020.140451\u003c/li\u003e\n\u003cli\u003eMcGlathery KJ, Pedersen MF, Borum J (1996) Changes in intracellular nitrogen pools and feedback controls on nitrogen uptake in Chaetomorpha linum (Chlorophyta) 1. Journal of Phycology 32 (3):393-401\u003c/li\u003e\n\u003cli\u003eMendez RL, Kwon JY (2023) Precipitation and characterization of Pacific dulse (Devaleraea mollis) proteins. Journal of Applied Phycology 35 (1):301-315\u003c/li\u003e\n\u003cli\u003eMorgan KC, Wright JL, Simpson F (1980) Review of chemical constituents of the red alga Palmaria palmata (dulse). Economic Botany 34 (1):27-50\u003c/li\u003e\n\u003cli\u003eMouritsen OG, Dawczynski C, Duelund L, Jahreis G, Vetter W, Schr\u0026ouml;der M (2013) On the human consumption of the red seaweed dulse (Palmaria palmata (L.) Weber \u0026amp; Mohr). Journal of Applied Phycology 25:1777-1791\u003c/li\u003e\n\u003cli\u003eNaldi M, Wheeler PA (1999) Changes in nitrogen pools in Ulva fenestrata (Chlorophyta) and Gracilaria pacifica (Rhodophyta) under nitrate and ammonium enrichment. Journal of Phycology 35 (1):70-77\u003c/li\u003e\n\u003cli\u003eNaylor RL, Hardy RW, Buschmann AH, Bush SR, Cao L, Klinger DH, Little DC, Lubchenco J, Shumway SE, Troell M (2021) A 20-year retrospective review of global aquaculture. Nature 591 (7851):551-563\u003c/li\u003e\n\u003cli\u003eNishihara GN, Terada R, Noro T (2005) Effect of temperature and irradiance on the uptake of ammonium and nitrate by Laurencia brongniartii (Rhodophyta, Ceramiales). Journal of Applied Phycology 17:371-377\u003c/li\u003e\n\u003cli\u003eOtt FD (1965) Synthetic media and techniques for the xenic cultivation of marine algae and flagellate. Va J Sci 16:205-218\u003c/li\u003e\n\u003cli\u003ePereira R, Yarish C, Critchley AT (2024) Seaweed aquaculture for human foods in land based and IMTA systems. In: Applications of Seaweeds in Food and Nutrition. Elsevier, pp 77-99\u003c/li\u003e\n\u003cli\u003ePorse H, Rudolph B (2017) The seaweed hydrocolloid industry: 2016 updates, requirements, and outlook. Journal of applied phycology 29 (5):2187-2200\u003c/li\u003e\n\u003cli\u003ePritchard DW, Hurd CL, Beardall J, Hepburn CD (2015) Restricted use of nitrate and a strong preference for ammonium reflects the nitrogen ecophysiology of a light‐limited red alga. Journal of phycology 51 (2):277-287\u003c/li\u003e\n\u003cli\u003eRedmond S, Green L, Yarish C, Kim J, Neefus C (2014) New England seaweed culture handbook: nursery systems.\u003c/li\u003e\n\u003cli\u003eRizzo NR, Beckert SB, Boles SE, Gross JA (2024) Temperature-induced variations in dulse (Devaleraea mollis) nutrition provide indirect benefits on juvenile red abalone (Haliotis rufescens) Growth. Frontiers in Marine Science 11. doi:10.3389/fmars.2024.1336793\u003c/li\u003e\n\u003cli\u003eRoleda MY, Hurd CL (2019) Seaweed nutrient physiology: application of concepts to aquaculture and bioremediation. Phycologia 58 (5):552-562. doi:10.1080/00318884.2019.1622920\u003c/li\u003e\n\u003cli\u003eSaunders GW, Jackson C, Salomaki ED (2018) Phylogenetic analyses of transcriptome data resolve familial assignments for genera of the red-algal Acrochaetiales-Palmariales Complex (Nemaliophycidae). Molecular phylogenetics and evolution 119:151-159\u003c/li\u003e\n\u003cli\u003eSchmedes PS, Nielsen MM (2020) Productivity and growth rate in Palmaria palmata affected by salinity, irradiance, and nutrient availability\u0026mdash;the use of nutrient pulses and interventional cultivation. Journal of Applied Phycology 32 (6):4099-4111\u003c/li\u003e\n\u003cli\u003eShannon E, Abu-Ghannam N (2019) Seaweeds as nutraceuticals for health and nutrition. Phycologia 58 (5):563-577. doi:10.1080/00318884.2019.1640533\u003c/li\u003e\n\u003cli\u003eShin SK, Kim S-K, Kim J-H, Han T, Yarish C, Kim JK (2020) Effects of stocking density on the productivity and nutrient removal of Agarophyton vermiculophyllum in Paralichthys olivaceus biofloc effluent. Journal of Applied Phycology 32:2605-2614\u003c/li\u003e\n\u003cli\u003eSingh S, Parihar P, Singh R, Singh VP, Prasad SM (2016) Heavy metal tolerance in plants: role of transcriptomics, proteomics, metabolomics, and ionomics. Frontiers in plant science 6:165395\u003c/li\u003e\n\u003cli\u003eSkriptsova A, Kalita T (2020) A re-evaluation of Palmaria (Palmariaceae, Rhodophyta) in the Northwest Pacific. European Journal of Phycology 55 (3):266-274\u003c/li\u003e\n\u003cli\u003eSkriptsova AV, Suzuki M, Semenchenko AA (2022) Morphological variation in Northwest Pacific Devaleraea mollis and description of D. inkyuleei sp. nov.(Palmariaceae, Rhodophyta). Phycologia 61 (6):606-615\u003c/li\u003e\n\u003cli\u003eSmith VH, Schindler DW (2009) Eutrophication science: where do we go from here? Trends in ecology \u0026amp; evolution 24 (4):201-207\u003c/li\u003e\n\u003cli\u003eSong H-I, Han S, Park J-S, Kim Y-J, Jeong C-B, Yarish C, Kim JK (2022) Dark treatment effect on the carrageenan characterization in a red alga, Chondrus crispus. Algal Research 68:102889\u003c/li\u003e\n\u003cli\u003eSt\u0026eacute;vant P, Schmedes PS, Le Gall L, Wegeberg S, Dumay J, Rebours C (2023) Concise review of the red macroalga dulse, Palmaria palmata (L.) Weber \u0026amp; Mohr. Journal of Applied Phycology 35 (2):523-550\u003c/li\u003e\n\u003cli\u003eSultana F, Wahab MA, Nahiduzzaman M, Mohiuddin M, Iqbal MZ, Shakil A, Mamun A-A, Khan MSR, Wong L, Asaduzzaman M (2023) Seaweed farming for food and nutritional security, climate change mitigation and adaptation, and women empowerment: A review. Aquaculture and Fisheries 8 (5):463-480. doi:https://doi.org/10.1016/j.aaf.2022.09.001\u003c/li\u003e\n\u003cli\u003eSuthar P, Gajaria TK, Reddy CRK (2019) Production of quality seaweed biomass through nutrient optimization for the sustainable land-based cultivation. Algal Research 42:101583. doi:https://doi.org/10.1016/j.algal.2019.101583\u003c/li\u003e\n\u003cli\u003eTitlyanov EA, Titlyanova TV (2010) Seaweed cultivation: Methods and problems. Russian Journal of Marine Biology 36 (4):227-242. doi:10.1134/S1063074010040012\u003c/li\u003e\n\u003cli\u003eVasconcelos M, Marson GV, Turgeon S, Tamigneaux E, Beaulieu L (2022) Environmental conditions influence on the physicochemical properties of wild and cultivated Palmaria palmata in the Canadian Atlantic shore. Journal of Applied Phycology 34 (5):2565-2578\u003c/li\u003e\n\u003cli\u003eWang Y, Feng Y, Liu X, Zhong M, Chen W, Wang F, Du H (2018) Response of Gracilaria lemaneiformis to nitrogen deprivation. Algal research 34:82-96\u003c/li\u003e\n\u003cli\u003eWellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. Journal of plant physiology 144 (3):307-313\u003c/li\u003e\n\u003cli\u003eWerner A, Dring M (2011) Aquaculture explained no. 27-cultivating Palmaria palmata. Bord Iascaigh Mhara 76\u003c/li\u003e\n\u003cli\u003eXu N, Xu K, Wang W, Xu Y, Ji D, Chen C, Xie C (2020) Nutrient enrichment improves growth and food quality of two strains of the economic seaweed Pyropia haitanensis. Frontiers in Marine Science 7:544582\u003c/li\u003e\n\u003cli\u003eYong WTL, Thien VY, Misson M, Chin GJWL, Said Hussin SNI, Chong HLH, Yusof NA, Ma NL, Rodrigues KF (2024) Seaweed: A bioindustrial game-changer for the green revolution. Biomass and Bioenergy 183:107122. doi:https://doi.org/10.1016/j.biombioe.2024.107122\u003c/li\u003e\n\u003cli\u003eYoung EB, Dring MJ, Savidge G, Birkett DA, Berges JA (2007) Seasonal variations in nitrate reductase activity and internal N pools in intertidal brown algae are correlated with ambient nitrate concentrations. Plant, cell \u0026amp; environment 30 (6):764-774\u003c/li\u003e\n\u003cli\u003eZhao L-S, Li K, Wang Q-M, Song X-Y, Su H-N, Xie B-B, Zhang X-Y, Huang F, Chen X-L, Zhou B-C (2017) Nitrogen starvation impacts the photosynthetic performance of Porphyridium cruentum as revealed by chlorophyll a fluorescence. Scientific Reports 7 (1):8542\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Devaleraea mollis, Palmaria hecatensis, Nutrient supplements, Growth, Pigment, Protein","lastPublishedDoi":"10.21203/rs.3.rs-4953297/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4953297/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eDevaleraea mollis\u003c/em\u003e and \u003cem\u003ePalmaria hecatensis\u003c/em\u003e have emerged as potential species for land-based cultivation of red seaweeds in the Pacific Northwest of the United States. Land-based cultivation has the advantage of customization of high-quality biomass production. However, the high material and preparation costs of the von Stosch enrichment medium (VSE) are a limitation of land-based cultivation of \u003cem\u003eD. mollis \u003c/em\u003eand \u003cem\u003eP. hecatensis\u003c/em\u003e. This study aims to reduce operational and management costs associated with controlling the culturing conditions of \u003cem\u003eD. mollis\u003c/em\u003e and \u003cem\u003eP. hecatensis\u003c/em\u003e without compromising biomass growth and quality in land-based tank cultivation systems. Five experimental treatments, 1) ambient seawater (AS); 2) VSE; 3) Guillard's f/2 medium (f/2); 4) commercial fertilizer, Jack's Special (JS); 5) JS with vitamin (JSV), were used in the present study. The growth, pigment, and protein content of \u003cem\u003eD. mollis \u003c/em\u003eand \u003cem\u003eP. hecatensis \u003c/em\u003ewere measured. Except for AS, \u003cem\u003ePalmaria hecatensis\u003c/em\u003e showed similar growth, pigment, and protein content at all experimental treatments. The growth and protein content of \u003cem\u003eD. mollis\u003c/em\u003eexposed to VSE were decreased by nitrogen limitation. However, the protein content of \u003cem\u003eD. mollis \u003c/em\u003eexposed to JS and JSV significantly increased without a decrease in growth. Therefore, the commercial fertilizer, Jack's Special (25-5-15), can replace the VSE for \u003cem\u003eD. mollis \u003c/em\u003eand \u003cem\u003eP. hecatensis\u003c/em\u003e, reducing operational and management costs link to nutrient supplementation.\u003c/p\u003e","manuscriptTitle":"Exploring Nutrient Supplements for Enhanced Growth and Quality of Devaleraea mollis and Palmaria hecatensis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-26 11:35:21","doi":"10.21203/rs.3.rs-4953297/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-03T21:09:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-23T00:44:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150600601323662633615738128821581380629","date":"2024-09-09T09:22:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-08T21:45:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60386003138173210293730023461420515036","date":"2024-09-01T21:02:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"89039501073252380262772001163277711184","date":"2024-08-29T23:43:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196665627520778944928351656257708105607","date":"2024-08-29T22:51:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-29T14:11:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-29T07:44:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-27T23:47:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Phycology","date":"2024-08-21T17:37:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"baf6483e-778d-4683-b066-78059b6a08e8","owner":[],"postedDate":"September 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-02-20T12:08:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-26 11:35:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4953297","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4953297","identity":"rs-4953297","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}