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

doi:10.21203/rs.3.rs-4953297/v1

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

https://doi.org/10.21203/rs.3.rs-4953297/v1

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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. mollisexposed 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

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 (NO₃⁻) and ammonium (NH₄⁺) 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.

2.1 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 Wt₂ and Wt₁ represent the weights of the thalli on days t₂ and t₁.

Table 1

Micro and macroelement concentration in different nutrient supplement
Nutrient (µM)	VSE	f/2	JS	JSV
Nitrogen	500 (100% NO₃^-)	500 (100% NO₃^-)	500 (57% NO₃^- and 43% NH₄⁺)	500 (57% NO₃^- and 43% NH₄⁺)
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 B₁ and B₁₂, Biotin	Vitamin B₁ and B₁₂, Biotin	-	Vitamin B₁ and B₁₂, 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.

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. 1A; 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. 1A; 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. 1A; 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. 1B; 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. 2B; 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. 2C; 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. 2E; 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. 2F; p = 0.014 and p < 0.001, respectively). Experimental treatments did not significantly influence tissue C content for either species (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 (NO₃⁻)	490.74 ± 0.02	432.72 ± 5.75	260.25 ± 1.94	259.18 ± 3.29
		Ammonium (NH₄⁺)	-	-	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 (NO₃⁻)	490.76 ± 0.02	433.24 ± 6.55	260.79 ± 2.46	258.91 ± 3.62
		Ammonium (NH₄⁺)	-	-	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 (NO₃⁻)	45.88 ± 7.92	66.87 ± 2.71	48.64 ± 6.70	45.58 ± 0.47
		Ammonium (NH₄⁺)	-	-	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 (NO₃⁻)	47.23 ± 8.86	69.18 ± 2.93	44.20 ± 8.25	47.44 ± 3.16
		Ammonium (NH₄⁺)	-	-	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. 4A; 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. 4A; 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. 4B; 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. 4B; 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. 5A; 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. 5A; 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. 5A; 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. 5B; p < 0.05). All treatments, except for AS, showed significantly higher protein content at day 14 versus day 0 of the experiment (Fig. 5B; 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 < 0.001), while the content in P. hecatensis was not (Fig. 6C and D; 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. 6A 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. 6A and B; p < 0.05).

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 (NO₂^-), 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.

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.

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).

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You are reading this latest preprint version

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

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Abstract

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1. Introduction

2. Materials and methods

2.1 Sample collection and pre-cultivation

2.2 Experimental treatments

2.3 Nutrient analysis

2.4 Photosynthesis efficiency and chlorophyll-a

2.5 Total protein and phycobiliprotein

2.6 Statistical analysis

3. Results

4. Discussion

5. Conclusion

Declarations

Author Contribution

Acknowledgement

References

Additional Declarations

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