Impacts of aquaculture nutrient sources: ammonium uptake of commercially important eucheumatoids depends on phosphate levels 1

Impacts of aquaculture nutrient sources: ammonium uptake of commercially important eucheumatoids depends on phosphate levels 1 | 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 Impacts of aquaculture nutrient sources: ammonium uptake of commercially important eucheumatoids depends on phosphate levels 1 Bienson Ceasar Narvarte, Lourie Ann Hinaloc, Shienna Mae Gonzaga, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2914668/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Sep, 2023 Read the published version in Journal of Applied Phycology → Version 1 posted 7 You are reading this latest preprint version Abstract In an integrated multitrophic aquaculture (IMTA) system, seaweeds serve as extractive species that utilize excess nutrients thereby reducing the risk of eutrophication and promoting sustainable aquaculture. However, the use of excessive fish feeds and the resultant fecal waste as nutrient streams can contribute to variations in nitrogen and phosphorus levels (e.g., primarily NH 4 + and PO 4 -3 ) in the surrounding area, and this may impact the physiology of the integrated seaweeds particularly on how these species take up inorganic nutrients. In this study, the effect of different PO 4 -3 levels on NH 4 + uptake of the three commercially important eucheumatoids Kappaphycus alvarezii , Kappaphycus striatus and Eucheuma denticulatum was examined under laboratory conditions. Seaweed thalli (n = 4) were incubated in seawater media containing 30 µM NH 4 + , representing eutrophic conditions, and 0, 0.5, 1.0, 1.5, 3.0 or 5.0 µM PO 4 -3 for 1 h under a saturating light level of 116 ± 7.13 µmol photons m -2 s -1 inside a temperature-controlled laboratory. Species-specific responses to PO 4 -3 levels were observed. For K. alvarezii , maximum NH 4 + uptake (17.8 ± 1.6 µmol gDW -1 h -1 ) was observed at 0.5 µM PO 4 -3 and the uptake rate declined at higher PO 4 -3 levels. For K. striatus , the NH 4 + uptake increases with increasing PO 4 -3 levels, with maximum N-uptake (6.35 ± 0.9 µmol gDW -1 h -1 ) observed at 5.0 µM PO 4 -3 . For E. denticulatum, maximum NH 4 + uptake (14.6 ± 1.4 µmol gDW -1 h -1 ) was observed at 1.0 µM PO 4 -3 . Our results suggest that, among the three eucheumatoid species, the NH 4 + uptake of K. striatus persist even at high levels of PO 4 -3 . However, our results also showed that K. striatus had the lowest range of NH 4 + uptake rates. These results should be taken into consideration when incorporating eucheumatoids in IMTA system where PO 4 -3 levels significantly vary in space and time. Biofiltration Eucheuma Eutrophication IMTA Kappaphycus Figures Figure 1 Figure 2 Introduction Seaweeds require carbon dioxide (CO 2 ), water (H 2 O) and light to photosynthesize and produce organic compounds. In addition, seaweeds also require nutrients from the surrounding seawater to maintain their health and sustain their physiological and metabolic functions. Two of the most essential nutrients required by macroalgae are nitrogen (N) and phosphorus (P). Generally, macroalgae utilize inorganic nitrogen such as ammonium (NH 4 + ), nitrate (NO 3 − ) or nitrite (NO 2 − ), although some macroalgae can also use organic nitrogen-based compounds such as urea as their nitrogen source (Probyn and Chapman 1982 ; Phillips and Hurd 2004 ; Smith et al. 2021 ). On the other hand, seaweeds typically absorb phosphorus in its orthophosphate (PO 4 − 3 ) form. Due to the significant amounts of phosphorus and nitrogen that seaweeds require, these two elements are considered as macronutrients (Harrison and Hurd 2001 ). Moreover, these two are essential because they are used by macroalgae to synthesize biochemical compounds (e.g., pigments, proteins, amino acids, phospholipids, nucleotides, sugar-phosphates, etc.) that are required for their proper functioning (Douglas et al. 2014 ; Roleda and Hurd 2019 ). Other nutrients such as iron (Fe), zinc (Zn) and copper (Cu) are used in smaller quantities and therefore regarded as micronutrients (Harrison and Hurd 2001 ). The ability of macroalgae to take up and assimilate inorganic nutrients from the seawater makes them a good candidate extractive species in an integrated multi-trophic aquaculture (IMTA). In this set-up, the excess nutrients are absorbed by seaweeds leading to lowered eutrophication risks while concurrently promoting sustainable aquaculture (Chopin et al. 2001 ). However, not all seaweeds can be incorporated into an IMTA system. The eco-physiological characteristics and market value of the candidate seaweed must always be taken into consideration when incorporating it in a seaweed-based integrated aquaculture (Kang et al. 2013 ). Seaweeds to be integrated in an IMTA system must have fast growth rates and high nutrient uptake rates to effectively remove and assimilate nutrients from the mariculture effluents (Chopin et al. 2001 ; Neori et al. 2004 ; Kang et al. 2013 ). The integrated seaweeds should maintain good health for considerable periods of time and withstand various types of environmental stresses that can be encountered during cultivation (Neori et al. 2004 ; Kang et al. 2013 ). Lastly, seaweeds in an IMTA system should be easy to cultivate and have high demand and market value (Buschmann et al. 1996 ; Neori et al. 2004 ). The growth and efficiency of seaweeds as biofilters in an IMTA are dependent on several culture conditions (Buschmann et al. 2001 ). For instance, seaweeds in an IMTA system are subject to fluctuating nutrient levels. In mariculture farms, the variability of nutrient levels can result from different waste streams, e.g., excretion of reared organisms, direct enrichment by applied feeds, and remineralization through microbial degradation of organic compounds (Burford and Williams 2001 ; Bouwman et al. 2013 ). In Bolinao-Anda, Pangasinan, Philippines, the degradation of uneaten fish feeds in an intensive mariculture site had resulted to high P levels (up to 4 µM during dry season) and sustained eutrophic conditions around the area (Ferrera et al. 2016 ). The impacts of high P levels on invertebrates (e.g., Uddin et al. 2016 ), corals (e.g., Klinges et al. 2022 ; Mezger et al. 2022 ), and phytoplankton (e.g., Smith 2006 ; Eker-Develi et al. 2006 ) had been thoroughly investigated. On the contrary, studies on how PO 4 − 3 affects the physiology of commercially important seaweeds are limited. Specifically, little information is available on how elevated PO 4 − 3 concentrations affect the uptake of other nutrients. This needs to be investigated because this may have significant implications when incorporating seaweeds in an IMTA system, where nutrient levels fluctuate both spatially and temporally. Eucheumatoids are a group of macroalgae that are considered good candidate species in an IMTA set-up due to their biofiltration potentials and established market (Rodrigueza and Montaño 2007 ; Melendres 2021 ; Narvarte et al. 2022 ). These macroalgae are farmed globally due to their phycocolloid (carrageenan) content, which is used in a wide array of economic and industrial applications (Bixler and Porse 2011 ). Thus, the incorporation of eucheumatoids in an IMTA system can provide additional source of income for farmers (da Silva et al. 2022 ). The potentials of eucheumatoids as co-culture species in an IMTA system has been investigated in a number of studies (e.g., Hayashi et al. 2008 ; Kambey et al. 2020 ; Pires et al. 2021 ). However, these studies have only focused on the growth and instantaneous nutrient uptake performance of these seaweeds. In-depth research, e.g., mechanistic response with ecological and economic implications, should also be done on the nutrient uptake responses of commercially important eucheumatoids to environmental changes observed in an IMTA set up, like fluctuating nutrient levels. The objective of this study is to evaluate the effects of different PO 4 − 3 levels on the NH 4 + uptake of commercially important carragenophytes Kappaphycus alvarezii , Kappaphycus striatus and Eucheuma denticulatum . The uptake rates were determined by incubating samples under different concentrations of PO 4 − 3 under laboratory conditions and measuring the amount of NH 4 + absorbed by seaweed samples. The results of our study have important implications on selecting which species are to be incorporated in an IMTA. Materials and Methods Collection, identification, acclimatization of eucheumatoids Samples of K. alvarezii var. tambalang , K. striatus var. sacol and E. denticulatum var. spinosum (Fig. 1 ) were obtained from the land-based hatchery cultures of Algal Ecophysiology Laboratory (Algae Lab) located in the University of the Philippines Marine Science Institute- Bolinao Marine Laboraory (UPMSI-BML), Bolinao, Pangasinan, Philippines. Healthy samples, characterized by the absence of ice-ice disease and epiphyte infestation, were collected using sterile blades. All seaweed samples were previously identified using DNA barcoding (Roleda et al. 2021 ). The collected samples were acclimated, given sufficient time to heal wounds, and then starved overnight in a nutrient-deplete artificial seawater (ASW, prepared by dissolving commercial sea salt in distilled water as instructed by the manufacturer) inside a temperature controlled (25°C) laboratory. The samples were placed in a transparent jar, bubbled with air to maintain water movement, and exposed to 116 ± 7.13 µmol photons m − 2 s − 1 irradiance. Preparation of incubation media Nutrient-deplete natural seawater (NDSW) was used in the preparation of experimental media. NDSW was prepared by stripping inorganic nutrients (NH 4 + , NO 3 − , NO 2 − and PO 4 − 3 ) from natural, UV- sterilized seawater. Approximately, 470 g of clean Ulva sp. (blade) was incubated in a 20 L tank filled with natural seawater inside a temperature-controlled room (25°C) under 147 ± 4.27 µmol photons m − 2 s − 1 irradiance provided by 4 × 20 W daylight LED lamps (LT8S-20W-DL, Omni, Manila, Philippines). The tank was also continuously bubbled with air to reduce the diffusion boundary layer, thereby enhancing the nutrient uptake rate of Ulva sp. After 18 h of incubation, the seawater was filtered using a 200 µm polyester filter bag, and seawater samples were obtained to measure the inorganic nutrient concentrations spectrophotometrically. All nutrients were found to have undetectable concentrations. The media for the experiment proper consisted of seawater with 6 different PO 4 − 3 levels (0, 0.5, 1.0, 1.5, 3.0 and 5.0 µM PO 4 − 3 ) and a constant NH 4 + concentration (30 µM representing eutrophic conditions). These media were prepared by dissolving appropriate amount of potassium dihydrogen phosphate (KH 2 PO 4 ) and ammonium chloride (NH 4 Cl), respectively, in NDSW. NH 4 + uptake of eucheumatoids To measure the NH 4 + uptake rates of eucheumatoids at different PO 4 − 3 levels, 2–3 g of fresh seaweed samples (n = 4 for each species) were incubated in an Erlenmeyer flask containing 70 ml of each of the above-mentioned media. The incubation was carried out inside a temperature-controlled (25°C) laboratory with a saturating irradiance of 116 ± 7.13 µmol photons m − 2 s − 1 (measured using LiCOR, LI-1400 light meter with cosine sensor) supplied by 3 × 20 W daylight LED lamps (LT8S-20W-DL, Omni, Manila, Philippines). All experimental units were haphazardly placed in an orbital shaker (KJ201BD, Wincom Company Ltd, Changsha Hunan, China) set at 140 rpm to reduce the boundary layer. A blank sample (i.e., experimental unit without the seaweed sample) was also incubated to determine uptake other than that caused by the eucheumatoid samples. After 1 h incubation, 10 mL of the seawater was pipetted, stored in 15 mL polyethylene centrifuge tubes, and analyzed for remaining NH 4 + ions. The analysis was done spectrophotometrically using 1240 mini UV-Vis spectrophotometer (Shimadzu, Japan) following the standard methods of Strickland and Parsons ( 1972 ). After the experiment, all seaweed samples were dried inside a 60°C oven until a constant dry weight (DW) was obtained. Data analyses The percent reduction in NH 4 + concentration was calculated based on the difference in NH 4 + concentration prior to and after the incubation. The uptake rate was calculated using the formula: $$V=\frac{\left({\text{N}}_{i}-{\text{N}}_{\text{f}}\right) \times \text{v}\text{o}\text{l}}{\text{D}\text{W} \times t}$$ Here, N i is the initial NH 4 + concentration, N f is the concentration of the remaining NH 4 + after incubation, ‘vol’ is the volume of seawater, DW is the dry weight of the seaweed sample and t is the duration of incubation. All NH 4 + uptake data were expressed as mean ± standard error (mean ± SE). For each species, significant variations in NH 4 + uptake rates among different PO 4 -3 levels were assessed using one-way analysis of variance (ANOVA). This was followed by Tukey’s post hoc test when significant variations were observed. The ANOVA was performed after the normality (Shapiro-Wilk test) and homoscedasticity (Levine’s test) of data were satisfied. All significance levels were set at α = 0.05. All statistical analyses and data visualization were done using the software SPSS v23 (IBM Corp). Results The NH 4 + concentration in the blank samples did not significantly vary before and after the incubation period. Therefore, the decrease in NH 4 + concentration in all other experimental units can only be attributed to the uptake of the seaweed samples. All three eucheumatoid species were able to reduce the amount of NH 4 + in the medium under different PO 4 − 3 levels. K. alvarezii , K. striatus and E. denticulatum were able to reduce the NH 4 + concentration by 40–66%, 21–59%, and 39–59%, respectively. The NH 4 + uptake rates of the three eucheumatoid species under different PO 4 − 3 levels were observed to be species-specific (Table 1 and Fig. 2 ). Among the three eucheumatoid species, K. striatus had the lowest range of NH 4 + uptake rate. This is followed by E. denticulatum and K. alvarezii (Table 1 ). Table 1 Range of NH 4 + uptake (n = 4) of the 3 eucheumatoid species under laboratory conditions Eucheumatoid species NH 4 + uptake rate, µmol gDW − 1 h − 1 Kappaphycus alvarezii 8.36–17.8 Kappaphycus striatus 3.36–6.35 Eucheuma denticulatum 6.60–14.6 Interestingly, all eucheumatoid species had a positive NH 4 + uptake rate even at 0 µM PO 4 − 3 level in the medium (Fig. 2 ). The NH 4 + uptake rates at 0 µM PO 4 − 3 were also significantly different among the three species examined (one-way ANOVA, F 2,9 = 32.9, p < 0.05). The NH 4 + uptake rate at 0 µM PO 4 − 3 of K. striatus (3.43 ± 0.77 µmol gDW − 1 h − 1 ) was significantly lower compared to that of K. alvarezii (12.7 ± 0.99 µmol gDW − 1 h − 1 , Tukey’s post hoc, p < 0.05) and E. denticulatum (9.71 ± 0.66 µmol gDW − 1 h − 1 , Tukey’s post hoc, p = 0.001). However, no significant difference was observed between the NH 4 + uptake rate at 0 µM PO 4 − 3 of K. alvarezii and E. denticulatum (Tukey’s post hoc, p = 0.075). The NH 4 + uptake rate of K. alvarezii varied among the PO 4 − 3 levels (Fig. 2 a, one-way ANOVA, F 5,18 = 16.3, p < 0.05). The maximum NH 4 + uptake rate observed at 0.5 µM PO 4 − 3 (17.8 ± 1.6 µmol gDW − 1 h − 1 ) was comparable to the NH 4 + uptake rate at 1.0 (14.5 ± 0.64 µmol gDW − 1 h − 1 ; Tukey’s post hoc, p = 0.717) and 1.5 µM PO 4 − 3 (13.6 ± 0.79 µmol gDW − 1 h − 1 ; Tukey’s post hoc, P = 0.970). However, at PO 4 − 3 levels ≥ 3.0 µM, the NH 4 + uptake rate significantly dropped by 52–53% (Fig. 2 a; Tukey’s post hoc, p > 0.05). The NH 4 + uptake rate of K. striatus increased with increasing PO 4 − 3 level, although the increase was not statistically significant (Fig. 2 b; one way ANOVA, F 5,18 = 2.87, p > 0.05). The lowest NH 4 + uptake rate (3.43 ± 0.77 µmol gDW − 1 h − 1 ) was observed at 0 µM PO 4 − 3 while the highest NH 4 + uptake (6.35 ± 1.0 µmol gDW − 1 h − 1 ) was observed at 5.0 µM PO 4 − 3 . For E. denticulatum , the NH 4 + uptake rates significantly varied among the PO 4 − 3 levels (Fig. 2 c; one way ANOVA, F 5,18 = 4.18, p < 0.05). The maximum uptake rate (14.6 ± 1.4 µmol gDW − 1 h − 1 ) observed at 1.0 µM PO 4 − 3 was statistically higher than the uptake rates at 0 and 0.5 µM PO 4 − 3 . At PO 4 − 3 levels ≥ 1.5 µM, the NH 4 + uptake of E. denticulatum significantly decreased by 40–43% (Fig. 2 c, Tukey’s post hoc, p < 0.05). Discussion The three eucheumatoid species examined in our study were able to significantly reduce NH 4 + concentration under different PO 4 − 3 levels. This corroborates the studies of other authors (e.g., Rodrigueza and Montaño 2007 ; Hayashi et al. 2008 ; Narvarte et al. 2022 ) who demonstrated the efficiency of eucheumatoids in absorbing NH 4 + ions. Furthermore, K. alvarezii and E. denticulatum had also been shown to be efficient in absorbing other nutrients like PO 4 − 3 , NO 3 − and NO 2 − (Hayashi et al. 2008 ; Kambey et al. 2020 ). These findings, along with those of our current study, indicate that the use of eucheumatoids as biofilters in an IMTA can successfully reduce the detrimental effects of eutrophication. The NH 4 + uptake rates of eucheumatoids under different PO 4 − 3 levels were species-specific, pointing to diverse ammonium uptake machineries found among eucheumatoids. Several ammonium transporters (AMTs) have been reported in seaweeds. For example, the chlorophyte Ulva linza possesses the AMT1, AMT2 and AMT3 subfamilies of ammonium transporters while the AMT1 subfamily predominates in the rhodophyte Pyropia yezoensis (Li et al. 2019 ; Fan et al. 2020 ). These diverse AMTs could also occur in eucheumatoids which enables them to exhibit distinct responses to PO 4 − 3 levels. Our study also showed that among the three species examined, K. striatus had the lowest range of NH 4 + uptake rate. This difference might be attributable to the low surface area to volume (SA: V) ratio of the seaweed samples used in the incubation experiment (Fig. 1 ). Theoretically, seaweeds with high SA: V ratio would have faster nutrient uptake rates because nutrients are absorbed across the entire surface area of the seaweed thallus (Taylor et al 1998 , Rosenberg and Ramus 1984 ). This is also supported by several published literatures showing a strong positive correlation between nutrient (NH 4 + , NO 3 − , PO 4 − 3 ) uptake rate and SA: V ratio (e.g.: Littler and Littler 1980 ; Rosenberg and Ramus 1984 ; Taylor et al. 1998 ; Narvarte et al. 2022 ). All eucheumatoid species showed a positive NH 4 + uptake rate even when the medium has 0 µM PO 4 − 3 . This may imply that either the presence of PO 4 − 3 ion in the bulk water is not a requirement for the uptake of NH 4 + or there is still sufficient pool of reserve tissue P after the seaweed samples were starved overnight prior to the uptake experiment, if internal P is essential for N uptake. However, when the reserved nutrients in their internal pools were exhausted to cope with the NDSW overnight, then the positive NH 4 + uptake rates at 0 µM PO 4 − 3 may therefore have resulted from the passive transport of NH 4 + when the samples were exposed to high NH 4 + levels, filling in the formerly empty nutrient pools. The results of our study also showed that the NH 4 + uptake rate at 0 µM PO 4 − 3 varied among the three eucheumatoid species. The NH 4 + uptake rate of eucheumatoids increased with increasing PO 4 − 3 levels. However, a decline in NH 4 + uptake rate was observed for K. alvarezii and E. denticulatum at ≥ 3.0 and ≥ 1.0, respectively, µM PO 4 − 3 . Similarly, the nitrogen uptake of Gracilaria lamaneiformis markedly increased at high PO 4 − 3 levels (Xu et al. 2010 ). The increase in NH 4 + uptake with increasing PO 4 − 3 level may suggest that PO 4 − 3 ions are possibly being converted to energy sources such as ATP, which could then be used to power the higher assimilation of nitrogen (i.e., production of pigments, proteins, amino acids, and other N-based compounds). This would subsequently result to more NH 4 + ions that are passively transported across cells. Moreover, a high PO 4 − 3 level is known to promote the regeneration of ribulose-1-5-bisphosphate (RuBP) resulting in enhanced photosynthetic efficiency (Rao and Terry 1989 ). This fast photosynthetic rate would generally be accompanied by faster nutrient uptake rates (Suárez-Álvarez et al. 2012 ). On the other hand, the decline in the NH 4 + uptake rates at higher PO 4 − 3 levels observed in K. alvarezii and E. denticulatum may indicate that high PO 4 − 3 levels may have hindered the uptake of NH 4 + ions. It is known that NH 4 + is primarily passively absorbed through transport proteins (i.e., through facilitated diffusion; Ninnemann et al. 1994 ; Hurd et al. 2014 ). Thus, PO 4 − 3 ions may have blocked these transport proteins preventing the uptake of NH 4 + . Alternatively, the serine residues of the transport proteins might have been phosphorylated at high PO 4 − 3 levels. In terrestrial plants, the phosphorylation of serine residues is known to inhibit the activity of the enzyme nitrate reductase (Bachmann et al. 1996 ; Lillo et al. 2004 ; Grossman and Aksoy 2015 ). The same mechanism might also be at work in the transport proteins of seaweeds. At high levels of PO 4 − 3 , the transport proteins of K. alvarezii and E. denticulatum might have been phosphorylated and this subsequently triggered conformational changes in its protein structure, thereby preventing the uptake of NH 4 + ions. In general, a high nutrient level tends to improve growth and nutrient uptake in seaweeds. For instance, K. alvarezii had higher growth and nutrient uptake at high levels of nutrient (Luhan et al. 2015 ; Narvarte et al. 2022 ). Moreover, Fucus vesiculosus samples that were enriched with N displayed the highest P uptake efficiency at biologically relevant P levels (Perini and Bracken 2014 ). Nevertheless, surpassing the optimal nutrient level might lead to adverse effects. In our study, the reduced NH 4 + uptake at high PO 4 − 3 levels observed in K. alvarezii and E. denticulatum might indicate that the seaweeds were being negatively impacted by the toxic level of PO 4 − 3 in the medium. Conversely, elevated PO 4 − 3 levels did not affect the NH 4 + uptake of K. striatus , suggesting that this species had unique N requirement. Rodrigueza and Montaño ( 2007 ) suggested that N assimilation in K. striatus was concentrated not only toward the synthesis of cell wall structures but also toward the formation of protoplasmic constituents. Thus, the uninhibited N uptake of K. striatus at high PO 4 − 3 level might imply that this species requires more N to synthesize such cellular components. The maximum NH 4 + uptake rate of the three eucheumatoid species also varied among the PO 4 − 3 levels. K. alvar ezii had maximum NH 4 + uptake at 0.5 µM PO 4 − 3 while K. striatus and E. denticulatum had maximum NH 4 + uptake rate at 5.0 and 1.0 µM PO 4 − 3 , respectively. This observation may reflect the nutrient requirements of seaweeds and their ability to respond with varying PO 4 − 3 levels, both of which are known to vary from species to species. In addition, these results have significant implications when incorporating these macroalgae in an IMTA system. Our results suggest that the uptake machineries of K. striatus are more tolerant to high PO 4 − 3 levels compared to that of K. alvarezii and E. denticulatum . However, our results also showed that K. striatus had the lowest range of NH 4 + uptake (Table 1 ) among the three eucheumatoid species, suggesting that the overnight starvation may have not been enough to empty the internal N pool and that K. striatus might have lots of nutrient reserves. This can be verified by e.g., analyzing the tissue N and P contents. For Ulva lactuca , it has been shown that the internal nutrient (N and P) storage was sufficient for approximately 10 days (Lubsch and Timmermans 2018 ). On the other hand, the internal storage capacity of eucheumatoids is yet to be investigated. Although the data presented in our study should be taken into consideration when incorporating eucheumatoids in IMTA, the results of our present study should not be the sole criterion in selecting which species works best in an IMTA set-up. It is important to consider other factors such as growth rate, tolerance to environmental stress, resistance to diseases and pest, and biochemical performance, when choosing species to be integrated in IMTA. Declarations Acknowledgement The AlgaE Team would like to thank the Bolinao Marine Laboratory (BML) for providing venue to conduct our experiments. BCV Narvarte and MY Roleda acknowledge the Sea6 Energy Pvt. Ltd. for the sponsorship during the 24 th International Seaweed Symposium (ISS) held on February 19-24, 2023, at Hobart, Tasmania, Australia. Likewise, BCV Narvarte and LAR Hinaloc would like to thank the University of the Philippines- Office of the International Linkages (UP-OIL) for providing them with a travel grant to attend the aforementioned symposium. BCV Narvarte also acknowledges the Department of Science and Technology- Philippine Council for Agriculture, Aquatic, and Natural Resources Research and Development (DOST-PCAARRD) for his PhD Scholarship. Funding This study was funded by the program Resource Inventory, Valuation and Policy in Ecosystem Services under Threat (RE-INVEST): Project I- Resource Inventory and Assessment of the West Philippine Sea of the Philippine Department of Science and Technology- Philippine Council for Agriculture, Aquatic, and Natural Resources Research and Development (DOST-PCAARRD). This study was also partly funded by the University of the Philippines-Marine Science Institute (UP-MSI) inhouse research grant. Authorship Contribution Statement BCV Narvarte : Investigation, Data Curation, Formal analysis, Visualization, Writing-Original Draft. LAR Hinaloc : Investigation, Visualization, Writing – review & editing. SMC Gonzaga : Investigation, Writing – review & editing. MY Roleda : Project administration, Supervision, Methodology, Writing- Review & Editing. 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The American Naturalist 116:25–44. https://doi.org/10.1086/283610 Lubsch A, Timmermans K (2018) Uptake kinetics and storage capacity of dissolved inorganic phosphorus and corresponding N:P dynamics in Ulva lactuca (Chlorophyta). J Phycol 54:215–223. https://doi.org/10.1111/jpy.12612 Luhan MRJ, Avañcena SS, Mateo JP (2015) Effect of short-term immersion of Kappaphycus alvarezii (Doty) Doty in high nitrogen on the growth, nitrogen assimilation, carrageenan quality, and occurrence of “ice-ice” disease. J Appl Phycol 27:917–922. https://doi.org/10.1007/s10811-014-0365-8 Melendres AR (2021) Growth and absorption response of Eucheuma denticulatum (Burman) Collins & Hervey) to fish farm water quality condition. JAMB 10:178–186. https://doi.org/10.15406/jamb.2021.10.00319 Mezger SD, Klinke A, Tilstra A, et al (2022) The widely distributed soft coral Xenia umbellata exhibits high resistance against phosphate enrichment and temperature increase. Sci Rep 12:22135. https://doi.org/10.1038/s41598-022-26325-5 Narvarte BCV, Genovia TGT, Hinaloc LAR, Roleda MY (2022) Growth, nitrate uptake kinetics, and biofiltration potential of eucheumatoids with different thallus morphologies. J Phycol 28:12–21. https://doi.org/10.1111/jpy.13229 Neori A, Chopin T, Troell M, et al (2004) Integrated aquaculture: rationale, evolution and state of the art emphasizing seaweed biofiltration in modern mariculture. Aquaculture 231:361–391. https://doi.org/10.1016/j.aquaculture.2003.11.015 Ninnemann O, Jauniaux JC, Frommer WB (1994) Identification of a high affinity NH4+ transporter from plants. The EMBO Journal 13:3464–3471. https://doi.org/10.1002/j.1460-2075.1994.tb06652.x Perini V, Bracken MES (2014) Nitrogen availability limits phosphorus uptake in an intertidal macroalga. Oecologia 175:667–676. https://doi.org/10.1007/s00442-014-2914-x Phillips JC, Hurd CL (2004) Kinetics of nitrate, ammonium, and urea uptake by four intertidal seaweeds from New Zealand. J Phycol 40:534–545. https://doi.org/10.1111/j.1529-8817.2004.03157.x Pires CM, Bazzo GC, Barreto PLM, et al (2021) Cultivation of the red seaweed Kappaphycus alvarezii using biofloc effluent. J Appl Phycol 33:1047–1058. https://doi.org/10.1007/s10811-020-02335-6 Probyn TA, Chapman ARO (1982) Nitrogen uptake characteristics of Chordaria flagelliformis (Phaeophyta) in batch mode and continuous mode experiments. Mar Biol 71:129–133. https://doi.org/10.1007/BF00394620 Rao IM, Terry N (1989) Leaf phosphate status, photosynthesis, and carbon partitioning in sugar beet: I. Changes in growth, gas exchange, and calvin cycle enzymes. Plant Physiol 90:814–819. https://doi.org/10.1104/pp.90.3.814 Rodrigueza MRC, Montaño MNE (2007) Bioremediation potential of three carrageenophytes cultivated in tanks with seawater from fish farms. J Appl Phycol 19:755–762. https://doi.org/10.1007/s10811-007-9217-0 Roleda MY, Aguinaldo Z-ZA, Crisostomo BA, et al (2021) Discovery of novel haplotypes from wild populations of Kappaphycus (Gigartinales, Rhodophyta) in the Philippines. ALGAE 36:1–12. https://doi.org/10.4490/algae.2021.36.2.18 Roleda MY, Hurd CL (2019) Seaweed nutrient physiology: application of concepts to aquaculture and bioremediation. Phycologia 58:552–562. https://doi.org/10.1080/00318884.2019.1622920 Rosenberg G, Ramus J (1984) Uptake of inorganic nitrogen and seaweed surface area: volume ratios. Aquat Bot 19:65–72. https://doi.org/10.1016/0304-3770(84)90008-1 Smith JM, Blasco G, Brzezinski MA, et al (2021) Factors influencing urea use by giant kelp ( Macrocystis pyrifera , Phaeophyceae). Limnol Oceanogr 66:1190–1200. https://doi.org/10.1002/lno.11674 Smith VH (2006) Responses of estuarine and coastal marine phytoplankton to nitrogen and phosphorus enrichment. Limnol Oceanogr 51:377–384. https://doi.org/10.4319/lo.2006.51.1_part_2.0377 Strickland JDH, Parsons TR (1972) A Practical Handbook of Seawater Analysis. Ottawa: Supply and Services, Canada Suárez-Álvarez S, Gómez-Pinchetti JL, García-Reina G (2012) Effects of increased CO 2 levels on growth, photosynthesis, ammonium uptake and cell composition in the macroalga Hypnea spinella (Gigartinales, Rhodophyta). J Appl Phycol 24:815–823. https://doi.org/10.1007/s10811-011-9700-5 Taylor R, Peek J, Rees T (1998) Scaling of ammonium uptake by seaweeds to surface area:volume ratio:geographical variation and the role of uptake by passive diffusion. Mar Ecol Prog Ser 169:143–148. https://doi.org/10.3354/meps169143 Uddin MdH, Shahjahan Md, Ruhul Amin AKM, et al (2016) Impacts of organophosphate pesticide, sumithion on water quality and benthic invertebrates in aquaculture ponds. Aquaculture Reports 3:88–92. https://doi.org/10.1016/j.aqrep.2016.01.002 Xu Z, Zou D, Gao K (2010) Effects of elevated CO 2 and phosphorus supply on growth, photosynthesis and nutrient uptake in the marine macroalga Gracilaria lemaneiformis (Rhodophyta). botm 53:123–129. https://doi.org/10.1515/BOT.2010.012 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 14 Sep, 2023 Read the published version in Journal of Applied Phycology → Version 1 posted Editorial decision: Major revision 06 Jul, 2023 Reviews received at journal 01 Jul, 2023 Reviewers agreed at journal 19 May, 2023 Reviewers invited by journal 16 May, 2023 Editor assigned by journal 11 May, 2023 Submission checks completed at journal 11 May, 2023 First submitted to journal 10 May, 2023 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. 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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-2914668","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":199041775,"identity":"3b220e26-4a5e-4b01-8ba7-a80d26b11e8e","order_by":0,"name":"Bienson Ceasar Narvarte","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYBACAziLvYGBIQHEOMDAIMEAZePTIsHAc4BkLRIwNYS0mEs3P/vMU3Ovjl/ydfKHBwyH8/gOMB+8zcNwJw+XFss5x4xn8xwrlpCcnbsNaNHhYskDbMnWPAzPinE67EaCMeMMtgQJg9u524COOZy44QCPmTQPkNGAU0v6Z8YZ/xIk7G+e3fwBooX/GwEtOcYMH9uAtkjwbpCA2sKGV4vljJxiho99CZIzzoD8YpBeLHmYzdhyjsEznFrMJdI3MyR8S+Dnbz+7+eOPCus8vuPND2+8qbiDUwu6O4EhwAxmHCBOAwgkQGkStIyCUTAKRsFwBwBfr1vEr2rj1wAAAABJRU5ErkJggg==","orcid":"","institution":"Algal Ecophysiology Laboratory, The Marine Science Institute, University of the Philippines, Diliman","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Bienson","middleName":"Ceasar","lastName":"Narvarte","suffix":""},{"id":199041776,"identity":"f9495675-b631-4290-a89a-2e2416a6396e","order_by":1,"name":"Lourie Ann Hinaloc","email":"","orcid":"","institution":"Algal Ecophysiology Laboratory, The Marine Science Institute, University of the Philippines, Diliman","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lourie","middleName":"Ann","lastName":"Hinaloc","suffix":""},{"id":199041777,"identity":"19a999b0-d523-4605-b23d-b8fbdc6e9bc0","order_by":2,"name":"Shienna Mae Gonzaga","email":"","orcid":"","institution":"Algal Ecophysiology Laboratory, The Marine Science Institute, University of the Philippines, Diliman","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shienna","middleName":"Mae","lastName":"Gonzaga","suffix":""},{"id":199041778,"identity":"80f36704-adc9-4749-960b-39015970fd98","order_by":3,"name":"Michael Roleda","email":"","orcid":"","institution":"Algal Ecophysiology Laboratory, The Marine Science Institute, University of the Philippines, Diliman","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Roleda","suffix":""}],"badges":[],"createdAt":"2023-05-10 06:44:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2914668/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2914668/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10811-023-03073-1","type":"published","date":"2023-09-14T10:22:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":37038529,"identity":"1a3105a9-d587-4ca9-954a-678ea27d57f4","added_by":"auto","created_at":"2023-05-15 14:21:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":749001,"visible":true,"origin":"","legend":"\u003cp\u003eCultures of \u003cem\u003eK. alvarezii\u003c/em\u003e (left), \u003cem\u003eK. striatus\u003c/em\u003e(center) and \u003cem\u003eE. denticulatum\u003c/em\u003e (right) of AlgaE Laboratory in Bolinao, Pangasinan, Philippines. The photos above are the representative samples of the actual thalli used in the nutrient uptake experiment. Scale bar = 2.0 cm\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2914668/v1/7cad6c0806ec9e4d2c62d505.png"},{"id":37039624,"identity":"66fb5e28-cde4-4573-baff-0c1decfb300c","added_by":"auto","created_at":"2023-05-15 14:29:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":139463,"visible":true,"origin":"","legend":"\u003cp\u003eMean ±SE (n = 4) NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rates of the eucheumatoids \u003cem\u003eK. alvarezii\u003c/em\u003e (A), \u003cem\u003eK. striatus\u003c/em\u003e (B), and \u003cem\u003eE. denticulatum\u003c/em\u003e (C) under different PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e levels. The subscripts \u003cem\u003ea, b\u003c/em\u003e and \u003cem\u003ec\u003c/em\u003e represent significant variations in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake among PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e levels (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2914668/v1/26db08056e0b919602236980.png"},{"id":56657322,"identity":"56359f71-8ba6-4276-9f18-b1c7913996b9","added_by":"auto","created_at":"2024-05-17 10:22:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1487743,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2914668/v1/06221eee-2812-45ee-a412-4cb0ad57e6b6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eImpacts of aquaculture nutrient sources: ammonium uptake of commercially important eucheumatoids depends on phosphate levels \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSeaweeds require carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e), water (H\u003csub\u003e2\u003c/sub\u003eO) and light to photosynthesize and produce organic compounds. In addition, seaweeds also require nutrients from the surrounding seawater to maintain their health and sustain their physiological and metabolic functions. Two of the most essential nutrients required by macroalgae are nitrogen (N) and phosphorus (P). Generally, macroalgae utilize inorganic nitrogen such as ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e), nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) or nitrite (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), although some macroalgae can also use organic nitrogen-based compounds such as urea as their nitrogen source (Probyn and Chapman \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Phillips and Hurd \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Smith et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). On the other hand, seaweeds typically absorb phosphorus in its orthophosphate (PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) form. Due to the significant amounts of phosphorus and nitrogen that seaweeds require, these two elements are considered as macronutrients (Harrison and Hurd \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Moreover, these two are essential because they are used by macroalgae to synthesize biochemical compounds (e.g., pigments, proteins, amino acids, phospholipids, nucleotides, sugar-phosphates, etc.) that are required for their proper functioning (Douglas et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Roleda and Hurd \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Other nutrients such as iron (Fe), zinc (Zn) and copper (Cu) are used in smaller quantities and therefore regarded as micronutrients (Harrison and Hurd \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe ability of macroalgae to take up and assimilate inorganic nutrients from the seawater makes them a good candidate extractive species in an integrated multi-trophic aquaculture (IMTA). In this set-up, the excess nutrients are absorbed by seaweeds leading to lowered eutrophication risks while concurrently promoting sustainable aquaculture (Chopin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). However, not all seaweeds can be incorporated into an IMTA system. The eco-physiological characteristics and market value of the candidate seaweed must always be taken into consideration when incorporating it in a seaweed-based integrated aquaculture (Kang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Seaweeds to be integrated in an IMTA system must have fast growth rates and high nutrient uptake rates to effectively remove and assimilate nutrients from the mariculture effluents (Chopin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Neori et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The integrated seaweeds should maintain good health for considerable periods of time and withstand various types of environmental stresses that can be encountered during cultivation (Neori et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Lastly, seaweeds in an IMTA system should be easy to cultivate and have high demand and market value (Buschmann et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Neori et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe growth and efficiency of seaweeds as biofilters in an IMTA are dependent on several culture conditions (Buschmann et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). For instance, seaweeds in an IMTA system are subject to fluctuating nutrient levels. In mariculture farms, the variability of nutrient levels can result from different waste streams, e.g., excretion of reared organisms, direct enrichment by applied feeds, and remineralization through microbial degradation of organic compounds (Burford and Williams \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Bouwman et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In Bolinao-Anda, Pangasinan, Philippines, the degradation of uneaten fish feeds in an intensive mariculture site had resulted to high P levels (up to 4 \u0026micro;M during dry season) and sustained eutrophic conditions around the area (Ferrera et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The impacts of high P levels on invertebrates (e.g., Uddin et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), corals (e.g., Klinges et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mezger et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and phytoplankton (e.g., Smith \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Eker-Develi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) had been thoroughly investigated. On the contrary, studies on how PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e affects the physiology of commercially important seaweeds are limited. Specifically, little information is available on how elevated PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e concentrations affect the uptake of other nutrients. This needs to be investigated because this may have significant implications when incorporating seaweeds in an IMTA system, where nutrient levels fluctuate both spatially and temporally.\u003c/p\u003e \u003cp\u003eEucheumatoids are a group of macroalgae that are considered good candidate species in an IMTA set-up due to their biofiltration potentials and established market (Rodrigueza and Monta\u0026ntilde;o \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Melendres \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Narvarte et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These macroalgae are farmed globally due to their phycocolloid (carrageenan) content, which is used in a wide array of economic and industrial applications (Bixler and Porse \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Thus, the incorporation of eucheumatoids in an IMTA system can provide additional source of income for farmers (da Silva et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The potentials of eucheumatoids as co-culture species in an IMTA system has been investigated in a number of studies (e.g., Hayashi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kambey et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pires et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, these studies have only focused on the growth and instantaneous nutrient uptake performance of these seaweeds. In-depth research, e.g., mechanistic response with ecological and economic implications, should also be done on the nutrient uptake responses of commercially important eucheumatoids to environmental changes observed in an IMTA set up, like fluctuating nutrient levels.\u003c/p\u003e \u003cp\u003eThe objective of this study is to evaluate the effects of different PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels on the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake of commercially important carragenophytes \u003cem\u003eKappaphycus alvarezii\u003c/em\u003e, \u003cem\u003eKappaphycus striatus\u003c/em\u003e and \u003cem\u003eEucheuma denticulatum\u003c/em\u003e. The uptake rates were determined by incubating samples under different concentrations of PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e under laboratory conditions and measuring the amount of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e absorbed by seaweed samples. The results of our study have important implications on selecting which species are to be incorporated in an IMTA.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection, identification, acclimatization of eucheumatoids\u003c/h2\u003e \u003cp\u003eSamples of \u003cem\u003eK. alvarezii\u003c/em\u003e var. \u003cem\u003etambalang\u003c/em\u003e, \u003cem\u003eK. striatus\u003c/em\u003e var. \u003cem\u003esacol\u003c/em\u003e and \u003cem\u003eE. denticulatum\u003c/em\u003e var. \u003cem\u003espinosum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were obtained from the land-based hatchery cultures of Algal Ecophysiology Laboratory (Algae Lab) located in the University of the Philippines Marine Science Institute- Bolinao Marine Laboraory (UPMSI-BML), Bolinao, Pangasinan, Philippines. Healthy samples, characterized by the absence of ice-ice disease and epiphyte infestation, were collected using sterile blades. All seaweed samples were previously identified using DNA barcoding (Roleda et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The collected samples were acclimated, given sufficient time to heal wounds, and then starved overnight in a nutrient-deplete artificial seawater (ASW, prepared by dissolving commercial sea salt in distilled water as instructed by the manufacturer) inside a temperature controlled (25\u0026deg;C) laboratory. The samples were placed in a transparent jar, bubbled with air to maintain water movement, and exposed to 116\u0026thinsp;\u0026plusmn;\u0026thinsp;7.13 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e irradiance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of incubation media\u003c/h2\u003e \u003cp\u003eNutrient-deplete natural seawater (NDSW) was used in the preparation of experimental media. NDSW was prepared by stripping inorganic nutrients (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) from natural, UV- sterilized seawater. Approximately, 470 g of clean \u003cem\u003eUlva\u003c/em\u003e sp. (blade) was incubated in a 20 L tank filled with natural seawater inside a temperature-controlled room (25\u0026deg;C) under 147\u0026thinsp;\u0026plusmn;\u0026thinsp;4.27 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e irradiance provided by 4 \u0026times; 20 W daylight LED lamps (LT8S-20W-DL, Omni, Manila, Philippines). The tank was also continuously bubbled with air to reduce the diffusion boundary layer, thereby enhancing the nutrient uptake rate of \u003cem\u003eUlva\u003c/em\u003e sp. After 18 h of incubation, the seawater was filtered using a 200 \u0026micro;m polyester filter bag, and seawater samples were obtained to measure the inorganic nutrient concentrations spectrophotometrically. All nutrients were found to have undetectable concentrations.\u003c/p\u003e \u003cp\u003eThe media for the experiment proper consisted of seawater with 6 different PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels (0, 0.5, 1.0, 1.5, 3.0 and 5.0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) and a constant NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration (30 \u0026micro;M representing eutrophic conditions). These media were prepared by dissolving appropriate amount of potassium dihydrogen phosphate (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) and ammonium chloride (NH\u003csub\u003e4\u003c/sub\u003eCl), respectively, in NDSW.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake of eucheumatoids\u003c/h2\u003e \u003cp\u003eTo measure the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rates of eucheumatoids at different PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels, 2\u0026ndash;3 g of fresh seaweed samples (n\u0026thinsp;=\u0026thinsp;4 for each species) were incubated in an Erlenmeyer flask containing 70 ml of each of the above-mentioned media. The incubation was carried out inside a temperature-controlled (25\u0026deg;C) laboratory with a saturating irradiance of 116\u0026thinsp;\u0026plusmn;\u0026thinsp;7.13 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (measured using LiCOR, LI-1400 light meter with cosine sensor) supplied by 3 \u0026times; 20 W daylight LED lamps (LT8S-20W-DL, Omni, Manila, Philippines). All experimental units were haphazardly placed in an orbital shaker (KJ201BD, Wincom Company Ltd, Changsha Hunan, China) set at 140 rpm to reduce the boundary layer. A blank sample (i.e., experimental unit without the seaweed sample) was also incubated to determine uptake other than that caused by the eucheumatoid samples. After 1 h incubation, 10 mL of the seawater was pipetted, stored in 15 mL polyethylene centrifuge tubes, and analyzed for remaining NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e ions. The analysis was done spectrophotometrically using 1240 mini UV-Vis spectrophotometer (Shimadzu, Japan) following the standard methods of Strickland and Parsons (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). After the experiment, all seaweed samples were dried inside a 60\u0026deg;C oven until a constant dry weight (DW) was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData analyses\u003c/h2\u003e \u003cp\u003eThe percent reduction in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration was calculated based on the difference in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration prior to and after the incubation. The uptake rate was calculated using the formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$V=\\frac{\\left({\\text{N}}_{i}-{\\text{N}}_{\\text{f}}\\right) \\times \\text{v}\\text{o}\\text{l}}{\\text{D}\\text{W} \\times t}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eHere, N\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the initial NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration, N\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e is the concentration of the remaining NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e after incubation, \u0026lsquo;vol\u0026rsquo; is the volume of seawater, DW is the dry weight of the seaweed sample and \u003cem\u003et\u003c/em\u003e is the duration of incubation.\u003c/p\u003e \u003cp\u003eAll NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE). For each species, significant variations in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rates among different PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e levels were assessed using one-way analysis of variance (ANOVA). This was followed by Tukey\u0026rsquo;s post hoc test when significant variations were observed. The ANOVA was performed after the normality (Shapiro-Wilk test) and homoscedasticity (Levine\u0026rsquo;s test) of data were satisfied. All significance levels were set at α\u0026thinsp;=\u0026thinsp;0.05. All statistical analyses and data visualization were done using the software SPSS v23 (IBM Corp).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration in the blank samples did not significantly vary before and after the incubation period. Therefore, the decrease in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration in all other experimental units can only be attributed to the uptake of the seaweed samples.\u003c/p\u003e \u003cp\u003eAll three eucheumatoid species were able to reduce the amount of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in the medium under different PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels. \u003cem\u003eK. alvarezii\u003c/em\u003e, \u003cem\u003eK. striatus\u003c/em\u003e and \u003cem\u003eE. denticulatum\u003c/em\u003e were able to reduce the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration by 40\u0026ndash;66%, 21\u0026ndash;59%, and 39\u0026ndash;59%, respectively.\u003c/p\u003e \u003cp\u003eThe NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rates of the three eucheumatoid species under different PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels were observed to be species-specific (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among the three eucheumatoid species, \u003cem\u003eK. striatus\u003c/em\u003e had the lowest range of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate. This is followed by \u003cem\u003eE. denticulatum\u003c/em\u003e and \u003cem\u003eK. alvarezii\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\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\u003eRange of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake (n\u0026thinsp;=\u0026thinsp;4) of the 3 eucheumatoid species under laboratory conditions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEucheumatoid species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate, \u0026micro;mol gDW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eKappaphycus alvarezii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.36\u0026ndash;17.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eKappaphycus striatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.36\u0026ndash;6.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEucheuma denticulatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.60\u0026ndash;14.6\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\u003eInterestingly, all eucheumatoid species had a positive NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate even at 0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e level in the medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rates at 0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e were also significantly different among the three species examined (one-way ANOVA, F\u003csub\u003e2,9\u003c/sub\u003e = 32.9, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate at 0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e of \u003cem\u003eK. striatus\u003c/em\u003e (3.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77 \u0026micro;mol gDW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was significantly lower compared to that of \u003cem\u003eK. alvarezii\u003c/em\u003e (12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99 \u0026micro;mol gDW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Tukey\u0026rsquo;s post hoc, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and \u003cem\u003eE. denticulatum\u003c/em\u003e (9.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66 \u0026micro;mol gDW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Tukey\u0026rsquo;s post hoc, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). However, no significant difference was observed between the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate at 0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e of \u003cem\u003eK. alvarezii\u003c/em\u003e and \u003cem\u003eE. denticulatum\u003c/em\u003e (Tukey\u0026rsquo;s post hoc, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.075).\u003c/p\u003e \u003cp\u003eThe NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate of \u003cem\u003eK. alvarezii\u003c/em\u003e varied among the PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, one-way ANOVA, F\u003csub\u003e5,18\u003c/sub\u003e = 16.3, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The maximum NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate observed at 0.5 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 \u0026micro;mol gDW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was comparable to the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate at 1.0 (14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64 \u0026micro;mol gDW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Tukey\u0026rsquo;s post hoc, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.717) and 1.5 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79 \u0026micro;mol gDW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Tukey\u0026rsquo;s post hoc, P\u0026thinsp;=\u0026thinsp;0.970). However, at PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels\u0026thinsp;\u0026ge;\u0026thinsp;3.0 \u0026micro;M, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate significantly dropped by 52\u0026ndash;53% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea; Tukey\u0026rsquo;s post hoc, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate of \u003cem\u003eK. striatus\u003c/em\u003e increased with increasing PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e level, although the increase was not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb; one way ANOVA, F\u003csub\u003e5,18\u003c/sub\u003e = 2.87, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The lowest NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate (3.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77 \u0026micro;mol gDW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was observed at 0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e while the highest NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake (6.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 \u0026micro;mol gDW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was observed at 5.0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eE. denticulatum\u003c/em\u003e, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rates significantly varied among the PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec; one way ANOVA, F\u003csub\u003e5,18\u003c/sub\u003e = 4.18, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The maximum uptake rate (14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 \u0026micro;mol gDW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) observed at 1.0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e was statistically higher than the uptake rates at 0 and 0.5 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. At PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels\u0026thinsp;\u0026ge;\u0026thinsp;1.5 \u0026micro;M, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake of \u003cem\u003eE. denticulatum\u003c/em\u003e significantly decreased by 40\u0026ndash;43% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, Tukey\u0026rsquo;s post hoc, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe three eucheumatoid species examined in our study were able to significantly reduce NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration under different PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels. This corroborates the studies of other authors (e.g., Rodrigueza and Monta\u0026ntilde;o \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hayashi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Narvarte et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) who demonstrated the efficiency of eucheumatoids in absorbing NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e ions. Furthermore, \u003cem\u003eK. alvarezii\u003c/em\u003e and \u003cem\u003eE. denticulatum\u003c/em\u003e had also been shown to be efficient in absorbing other nutrients like PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (Hayashi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kambey et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These findings, along with those of our current study, indicate that the use of eucheumatoids as biofilters in an IMTA can successfully reduce the detrimental effects of eutrophication.\u003c/p\u003e \u003cp\u003eThe NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rates of eucheumatoids under different PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels were species-specific, pointing to diverse ammonium uptake machineries found among eucheumatoids. Several ammonium transporters (AMTs) have been reported in seaweeds. For example, the chlorophyte \u003cem\u003eUlva linza\u003c/em\u003e possesses the AMT1, AMT2 and AMT3 subfamilies of ammonium transporters while the AMT1 subfamily predominates in the rhodophyte \u003cem\u003ePyropia yezoensis\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Fan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These diverse AMTs could also occur in eucheumatoids which enables them to exhibit distinct responses to PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels. Our study also showed that among the three species examined, \u003cem\u003eK. striatus\u003c/em\u003e had the lowest range of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate. This difference might be attributable to the low surface area to volume (SA: V) ratio of the seaweed samples used in the incubation experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Theoretically, seaweeds with high SA: V ratio would have faster nutrient uptake rates because nutrients are absorbed across the entire surface area of the seaweed thallus (Taylor et al \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Rosenberg and Ramus \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). This is also supported by several published literatures showing a strong positive correlation between nutrient (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) uptake rate and SA: V ratio (e.g.: Littler and Littler \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Rosenberg and Ramus \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Taylor et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Narvarte et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll eucheumatoid species showed a positive NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate even when the medium has 0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. This may imply that either the presence of PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ion in the bulk water is not a requirement for the uptake of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e or there is still sufficient pool of reserve tissue P after the seaweed samples were starved overnight prior to the uptake experiment, if internal P is essential for N uptake. However, when the reserved nutrients in their internal pools were exhausted to cope with the NDSW overnight, then the positive NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rates at 0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e may therefore have resulted from the passive transport of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e when the samples were exposed to high NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e levels, filling in the formerly empty nutrient pools. The results of our study also showed that the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate at 0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e varied among the three eucheumatoid species.\u003c/p\u003e \u003cp\u003eThe NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate of eucheumatoids increased with increasing PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels. However, a decline in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate was observed for \u003cem\u003eK. alvarezii\u003c/em\u003e and \u003cem\u003eE. denticulatum\u003c/em\u003e at \u0026ge;\u0026thinsp;3.0 and \u0026ge;\u0026thinsp;1.0, respectively, \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. Similarly, the nitrogen uptake of \u003cem\u003eGracilaria lamaneiformis\u003c/em\u003e markedly increased at high PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels (Xu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The increase in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake with increasing PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e level may suggest that PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ions are possibly being converted to energy sources such as ATP, which could then be used to power the higher assimilation of nitrogen (i.e., production of pigments, proteins, amino acids, and other N-based compounds). This would subsequently result to more NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e ions that are passively transported across cells. Moreover, a high PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e level is known to promote the regeneration of ribulose-1-5-bisphosphate (RuBP) resulting in enhanced photosynthetic efficiency (Rao and Terry \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). This fast photosynthetic rate would generally be accompanied by faster nutrient uptake rates (Su\u0026aacute;rez-\u0026Aacute;lvarez et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). On the other hand, the decline in the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rates at higher PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels observed in \u003cem\u003eK. alvarezii\u003c/em\u003e and \u003cem\u003eE. denticulatum\u003c/em\u003e may indicate that high PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels may have hindered the uptake of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e ions. It is known that NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e is primarily passively absorbed through transport proteins (i.e., through facilitated diffusion; Ninnemann et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Hurd et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Thus, PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ions may have blocked these transport proteins preventing the uptake of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. Alternatively, the serine residues of the transport proteins might have been phosphorylated at high PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels. In terrestrial plants, the phosphorylation of serine residues is known to inhibit the activity of the enzyme nitrate reductase (Bachmann et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Lillo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Grossman and Aksoy \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The same mechanism might also be at work in the transport proteins of seaweeds. At high levels of PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, the transport proteins of \u003cem\u003eK. alvarezii\u003c/em\u003e and \u003cem\u003eE. denticulatum\u003c/em\u003e might have been phosphorylated and this subsequently triggered conformational changes in its protein structure, thereby preventing the uptake of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e ions.\u003c/p\u003e \u003cp\u003eIn general, a high nutrient level tends to improve growth and nutrient uptake in seaweeds. For instance, \u003cem\u003eK. alvarezii\u003c/em\u003e had higher growth and nutrient uptake at high levels of nutrient (Luhan et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Narvarte et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, \u003cem\u003eFucus vesiculosus\u003c/em\u003e samples that were enriched with N displayed the highest P uptake efficiency at biologically relevant P levels (Perini and Bracken \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Nevertheless, surpassing the optimal nutrient level might lead to adverse effects. In our study, the reduced NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake at high PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels observed in \u003cem\u003eK. alvarezii\u003c/em\u003e and \u003cem\u003eE. denticulatum\u003c/em\u003e might indicate that the seaweeds were being negatively impacted by the toxic level of PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e in the medium. Conversely, elevated PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels did not affect the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake of \u003cem\u003eK. striatus\u003c/em\u003e, suggesting that this species had unique N requirement. Rodrigueza and Monta\u0026ntilde;o (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) suggested that N assimilation in \u003cem\u003eK. striatus\u003c/em\u003e was concentrated not only toward the synthesis of cell wall structures but also toward the formation of protoplasmic constituents. Thus, the uninhibited N uptake of \u003cem\u003eK. striatus\u003c/em\u003e at high PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e level might imply that this species requires more N to synthesize such cellular components.\u003c/p\u003e \u003cp\u003eThe maximum NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate of the three eucheumatoid species also varied among the PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels. \u003cem\u003eK. alvar\u003c/em\u003eezii had maximum NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake at 0.5 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e while \u003cem\u003eK. striatus\u003c/em\u003e and \u003cem\u003eE. denticulatum\u003c/em\u003e had maximum NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rate at 5.0 and 1.0 \u0026micro;M PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, respectively. This observation may reflect the nutrient requirements of seaweeds and their ability to respond with varying PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels, both of which are known to vary from species to species. In addition, these results have significant implications when incorporating these macroalgae in an IMTA system. Our results suggest that the uptake machineries of \u003cem\u003eK. striatus\u003c/em\u003e are more tolerant to high PO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e levels compared to that of \u003cem\u003eK. alvarezii\u003c/em\u003e and \u003cem\u003eE. denticulatum\u003c/em\u003e. However, our results also showed that \u003cem\u003eK. striatus\u003c/em\u003e had the lowest range of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) among the three eucheumatoid species, suggesting that the overnight starvation may have not been enough to empty the internal N pool and that \u003cem\u003eK. striatus\u003c/em\u003e might have lots of nutrient reserves. This can be verified by e.g., analyzing the tissue N and P contents. For \u003cem\u003eUlva lactuca\u003c/em\u003e, it has been shown that the internal nutrient (N and P) storage was sufficient for approximately 10 days (Lubsch and Timmermans \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). On the other hand, the internal storage capacity of eucheumatoids is yet to be investigated. Although the data presented in our study should be taken into consideration when incorporating eucheumatoids in IMTA, the results of our present study should not be the sole criterion in selecting which species works best in an IMTA set-up. It is important to consider other factors such as growth rate, tolerance to environmental stress, resistance to diseases and pest, and biochemical performance, when choosing species to be integrated in IMTA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe AlgaE Team would like to thank the Bolinao Marine Laboratory (BML) for providing venue to conduct our experiments. BCV Narvarte and MY Roleda acknowledge the Sea6 Energy Pvt. Ltd. for the sponsorship during the 24\u003csup\u003eth\u003c/sup\u003e International Seaweed Symposium (ISS) held on February 19-24, 2023, at Hobart, Tasmania, Australia. Likewise, BCV Narvarte and LAR Hinaloc would like to thank the University of the Philippines- Office of the International Linkages (UP-OIL) for providing them with a travel grant to attend the aforementioned symposium. BCV Narvarte also acknowledges the Department of Science and Technology- Philippine Council for Agriculture, Aquatic, and Natural Resources Research and Development (DOST-PCAARRD) for his PhD Scholarship. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the program \u003cem\u003eResource Inventory, Valuation and Policy in Ecosystem Services under Threat (RE-INVEST): Project I- Resource Inventory and Assessment of the West Philippine Sea\u003c/em\u003e of the Philippine Department of Science and Technology- Philippine Council for Agriculture, Aquatic, and Natural Resources Research and Development (DOST-PCAARRD). This study was also partly funded by the University of the Philippines-Marine Science Institute (UP-MSI) inhouse research grant. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBCV Narvarte\u003c/strong\u003e: Investigation, Data Curation, Formal analysis, Visualization, Writing-Original Draft. \u003cstrong\u003eLAR Hinaloc\u003c/strong\u003e: Investigation, Visualization, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eSMC Gonzaga\u003c/strong\u003e: Investigation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eMY Roleda\u003c/strong\u003e: Project administration, Supervision, Methodology, Writing- Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBachmann M, Shiraishi N, Campbell WH, et al (1996) Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. Plant Cell 8:505\u0026ndash;517. https://doi.org/10.1105/tpc.8.3.505\u003c/li\u003e\n\u003cli\u003eBixler HJ, Porse H (2011) A decade of change in the seaweed hydrocolloids industry. J Appl Phycol 23:321\u0026ndash;335. https://doi.org/10.1007/s10811-010-9529-3\u003c/li\u003e\n\u003cli\u003eBouwman L, Beusen A, Glibert PM, et al (2013) Mariculture: significant and expanding cause of coastal nutrient enrichment. Environ Res Lett 8:044026. https://doi.org/10.1088/1748-9326/8/4/044026\u003c/li\u003e\n\u003cli\u003eBurford MA, Williams KC (2001) The fate of nitrogenous waste from shrimp feeding. Aquaculture 198:79\u0026ndash;93. https://doi.org/10.1016/S0044-8486(00)00589-5\u003c/li\u003e\n\u003cli\u003eBuschmann AH, Troell M, Kautsky N (2001) Integrated algal farming: a review. Cah Biol Mar 42:83\u0026ndash;90\u003c/li\u003e\n\u003cli\u003eBuschmann AH, Troell M, Kautsky N, Kautsky L (1996) Integrated tank cultivation of salmonids and \u003cem\u003eGracilaria chilensis\u003c/em\u003e (Gracilariales, Rhodophyta). Hydrobiologia 326\u0026ndash;327:75\u0026ndash;82. https://doi.org/10.1007/BF00047789\u003c/li\u003e\n\u003cli\u003eChopin T, Buschmann AH, Halling C, et al (2001) Integrating seaweeds into marine aquaculture systems: A key toward sustainability. Journal of Phycology 37:975\u0026ndash;986. https://doi.org/10.1046/j.1529-8817.2001.01137.x\u003c/li\u003e\n\u003cli\u003eda Silva EG, Castilho-Barros L, Henriques MB (2022) Economic feasibility of integrated multi-trophic aquaculture (mussel \u003cem\u003ePerna perna\u003c/em\u003e, scallop \u003cem\u003eNodipecten nodosus\u003c/em\u003e and seaweed \u003cem\u003eKappaphycus alvarezii\u003c/em\u003e) in Southeast Brazil: A small-scale aquaculture farm model. Aquaculture 552:738031. https://doi.org/10.1016/j.aquaculture.2022.738031\u003c/li\u003e\n\u003cli\u003eDouglas E, Haggitt T, Rees T (2014) Supply- and demand-driven phosphate uptake and tissue phosphorus in temperate seaweeds. Aquat Biol 23:49\u0026ndash;60. https://doi.org/10.3354/ab00601\u003c/li\u003e\n\u003cli\u003eEker-Develi E, Kideys AE, Tugrul S (2006) Effect of nutrients on culture dynamics of marine phytoplankton. Aquat Sci 68:28\u0026ndash;39. https://doi.org/10.1007/s00027-005-0810-5\u003c/li\u003e\n\u003cli\u003eFan X, Xu D, Wang D, et al (2020) Nutrient uptake and transporter gene expression of ammonium, nitrate, and phosphorus in \u003cem\u003eUlva linza\u003c/em\u003e: adaption to variable concentrations and temperatures. J Appl Phycol 32:1311\u0026ndash;1322. https://doi.org/10.1007/s10811-020-02050-2\u003c/li\u003e\n\u003cli\u003eFerrera CM, Watanabe A, Miyajima T, et al (2016) Phosphorus as a driver of nitrogen limitation and sustained eutrophic conditions in Bolinao and Anda, Philippines, a mariculture-impacted tropical coastal area. Mar Pollut Bull 105:237\u0026ndash;248. https://doi.org/10.1016/j.marpolbul.2016.02.025\u003c/li\u003e\n\u003cli\u003eGrossman AR, Aksoy M (2015) Algae in a phosphorus-limited landscape. In: Plaxton WC, Lambers H (eds) Annual Plant Reviews Volume 48. John Wiley \u0026amp; Sons, Inc., Hoboken, NJ, USA, pp 337\u0026ndash;374\u003c/li\u003e\n\u003cli\u003eHarrison PJ, Hurd CL (2001) Nutrient physiology of seaweeds: Application of concepts to aquaculture. Cah Biol Mar 42:71\u0026ndash;82\u003c/li\u003e\n\u003cli\u003eHayashi L, Yokoya NS, Ostini S, et al (2008) Nutrients removed by \u003cem\u003eKappaphycus alvarezii\u003c/em\u003e (Rhodophyta, Solieriaceae) in integrated cultivation with fishes in re-circulating water. Aquaculture 277:185\u0026ndash;191. https://doi.org/10.1016/j.aquaculture.2008.02.024\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\u003eKambey CSB, Sondak CFA, Chung I (2020) Potential growth and nutrient removal of \u003cem\u003eKappaphycus alvarezii\u003c/em\u003e in a fish floating‐net cage system in Sekotong Bay, Lombok, Indonesia. J World Aquacult Soc 51:944\u0026ndash;959. https://doi.org/10.1111/jwas.12683\u003c/li\u003e\n\u003cli\u003eKang YH, Hwang JR, Chung IK, Park SR (2013) Development of a seaweed species-selection index for successful culture in a seaweed-based integrated aquaculture system. J Ocean Univ China 12:125\u0026ndash;133. https://doi.org/10.1007/s11802-013-1928-z\u003c/li\u003e\n\u003cli\u003eKlinges JG, Patel SH, Duke WC, et al (2022) Phosphate enrichment induces increased dominance of the parasite \u003cem\u003eAquarickettsia\u003c/em\u003e in the coral \u003cem\u003eAcropora cervicornis\u003c/em\u003e. FEMS Microbiology Ecology 98:fiac013. https://doi.org/10.1093/femsec/fiac013\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 \u003cem\u003ePyropia yezoensis\u003c/em\u003e. AJPS 10:1325\u0026ndash;1349. https://doi.org/10.4236/ajps.2019.108096\u003c/li\u003e\n\u003cli\u003eLillo C, Meyer C, Lea US, et al (2004) Mechanism and importance of post-translational regulation of nitrate reductase. Journal of Experimental Botany 55:1275\u0026ndash;1282. https://doi.org/10.1093/jxb/erh132\u003c/li\u003e\n\u003cli\u003eLittler MM, Littler DS (1980) The evolution of thallus form and survival strategies in benthic marine macroalgae: field and laboratory tests of a functional form model. The American Naturalist 116:25\u0026ndash;44. https://doi.org/10.1086/283610\u003c/li\u003e\n\u003cli\u003eLubsch A, Timmermans K (2018) Uptake kinetics and storage capacity of dissolved inorganic phosphorus and corresponding N:P dynamics in \u003cem\u003eUlva lactuca\u003c/em\u003e (Chlorophyta). J Phycol 54:215\u0026ndash;223. https://doi.org/10.1111/jpy.12612\u003c/li\u003e\n\u003cli\u003eLuhan MRJ, Ava\u0026ntilde;cena SS, Mateo JP (2015) Effect of short-term immersion of Kappaphycus alvarezii (Doty) Doty in high nitrogen on the growth, nitrogen assimilation, carrageenan quality, and occurrence of \u0026ldquo;ice-ice\u0026rdquo; disease. J Appl Phycol 27:917\u0026ndash;922. https://doi.org/10.1007/s10811-014-0365-8\u003c/li\u003e\n\u003cli\u003eMelendres AR (2021) Growth and absorption response of \u003cem\u003eEucheuma denticulatum\u003c/em\u003e (Burman) Collins \u0026amp; Hervey) to fish farm water quality condition. JAMB 10:178\u0026ndash;186. https://doi.org/10.15406/jamb.2021.10.00319\u003c/li\u003e\n\u003cli\u003eMezger SD, Klinke A, Tilstra A, et al (2022) The widely distributed soft coral \u003cem\u003eXenia umbellata\u003c/em\u003e exhibits high resistance against phosphate enrichment and temperature increase. Sci Rep 12:22135. https://doi.org/10.1038/s41598-022-26325-5\u003c/li\u003e\n\u003cli\u003eNarvarte BCV, Genovia TGT, Hinaloc LAR, Roleda MY (2022) Growth, nitrate uptake kinetics, and biofiltration potential of eucheumatoids with different thallus morphologies. J Phycol 28:12\u0026ndash;21. https://doi.org/10.1111/jpy.13229\u003c/li\u003e\n\u003cli\u003eNeori A, Chopin T, Troell M, et al (2004) Integrated aquaculture: rationale, evolution and state of the art emphasizing seaweed biofiltration in modern mariculture. Aquaculture 231:361\u0026ndash;391. https://doi.org/10.1016/j.aquaculture.2003.11.015\u003c/li\u003e\n\u003cli\u003eNinnemann O, Jauniaux JC, Frommer WB (1994) Identification of a high affinity NH4+ transporter from plants. The EMBO Journal 13:3464\u0026ndash;3471. https://doi.org/10.1002/j.1460-2075.1994.tb06652.x\u003c/li\u003e\n\u003cli\u003ePerini V, Bracken MES (2014) Nitrogen availability limits phosphorus uptake in an intertidal macroalga. Oecologia 175:667\u0026ndash;676. https://doi.org/10.1007/s00442-014-2914-x\u003c/li\u003e\n\u003cli\u003ePhillips JC, Hurd CL (2004) Kinetics of nitrate, ammonium, and urea uptake by four intertidal seaweeds from New Zealand. J Phycol 40:534\u0026ndash;545. https://doi.org/10.1111/j.1529-8817.2004.03157.x\u003c/li\u003e\n\u003cli\u003ePires CM, Bazzo GC, Barreto PLM, et al (2021) Cultivation of the red seaweed \u003cem\u003eKappaphycus alvarezii\u003c/em\u003e using biofloc effluent. J Appl Phycol 33:1047\u0026ndash;1058. https://doi.org/10.1007/s10811-020-02335-6\u003c/li\u003e\n\u003cli\u003eProbyn TA, Chapman ARO (1982) Nitrogen uptake characteristics of \u003cem\u003eChordaria flagelliformis\u003c/em\u003e (Phaeophyta) in batch mode and continuous mode experiments. Mar Biol 71:129\u0026ndash;133. https://doi.org/10.1007/BF00394620\u003c/li\u003e\n\u003cli\u003eRao IM, Terry N (1989) Leaf phosphate status, photosynthesis, and carbon partitioning in sugar beet: I. Changes in growth, gas exchange, and calvin cycle enzymes. Plant Physiol 90:814\u0026ndash;819. https://doi.org/10.1104/pp.90.3.814\u003c/li\u003e\n\u003cli\u003eRodrigueza MRC, Monta\u0026ntilde;o MNE (2007) Bioremediation potential of three carrageenophytes cultivated in tanks with seawater from fish farms. J Appl Phycol 19:755\u0026ndash;762. https://doi.org/10.1007/s10811-007-9217-0\u003c/li\u003e\n\u003cli\u003eRoleda MY, Aguinaldo Z-ZA, Crisostomo BA, et al (2021) Discovery of novel haplotypes from wild populations of \u003cem\u003eKappaphycus\u003c/em\u003e (Gigartinales, Rhodophyta) in the Philippines. ALGAE 36:1\u0026ndash;12. https://doi.org/10.4490/algae.2021.36.2.18\u003c/li\u003e\n\u003cli\u003eRoleda MY, Hurd CL (2019) Seaweed nutrient physiology: application of concepts to aquaculture and bioremediation. Phycologia 58:552\u0026ndash;562. https://doi.org/10.1080/00318884.2019.1622920\u003c/li\u003e\n\u003cli\u003eRosenberg G, Ramus J (1984) Uptake of inorganic nitrogen and seaweed surface area: volume ratios. Aquat Bot 19:65\u0026ndash;72. https://doi.org/10.1016/0304-3770(84)90008-1\u003c/li\u003e\n\u003cli\u003eSmith JM, Blasco G, Brzezinski MA, et al (2021) Factors influencing urea use by giant kelp ( \u003cem\u003eMacrocystis pyrifera\u003c/em\u003e , Phaeophyceae). Limnol Oceanogr 66:1190\u0026ndash;1200. https://doi.org/10.1002/lno.11674\u003c/li\u003e\n\u003cli\u003eSmith VH (2006) Responses of estuarine and coastal marine phytoplankton to nitrogen and phosphorus enrichment. Limnol Oceanogr 51:377\u0026ndash;384. https://doi.org/10.4319/lo.2006.51.1_part_2.0377\u003c/li\u003e\n\u003cli\u003eStrickland JDH, Parsons TR (1972) A Practical Handbook of Seawater Analysis. Ottawa: Supply and Services, Canada\u003c/li\u003e\n\u003cli\u003eSu\u0026aacute;rez-\u0026Aacute;lvarez S, G\u0026oacute;mez-Pinchetti JL, Garc\u0026iacute;a-Reina G (2012) Effects of increased CO\u003csub\u003e2\u003c/sub\u003e levels on growth, photosynthesis, ammonium uptake and cell composition in the macroalga \u003cem\u003eHypnea spinella\u003c/em\u003e (Gigartinales, Rhodophyta). J Appl Phycol 24:815\u0026ndash;823. https://doi.org/10.1007/s10811-011-9700-5\u003c/li\u003e\n\u003cli\u003eTaylor R, Peek J, Rees T (1998) Scaling of ammonium uptake by seaweeds to surface area:volume ratio:geographical variation and the role of uptake by passive diffusion. Mar Ecol Prog Ser 169:143\u0026ndash;148. https://doi.org/10.3354/meps169143\u003c/li\u003e\n\u003cli\u003eUddin MdH, Shahjahan Md, Ruhul Amin AKM, et al (2016) Impacts of organophosphate pesticide, sumithion on water quality and benthic invertebrates in aquaculture ponds. Aquaculture Reports 3:88\u0026ndash;92. https://doi.org/10.1016/j.aqrep.2016.01.002\u003c/li\u003e\n\u003cli\u003eXu Z, Zou D, Gao K (2010) Effects of elevated CO \u003csub\u003e2\u003c/sub\u003e and phosphorus supply on growth, photosynthesis and nutrient uptake in the marine macroalga \u003cem\u003eGracilaria lemaneiformis\u003c/em\u003e (Rhodophyta). botm 53:123\u0026ndash;129. https://doi.org/10.1515/BOT.2010.012\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":"Biofiltration, Eucheuma, Eutrophication, IMTA, Kappaphycus","lastPublishedDoi":"10.21203/rs.3.rs-2914668/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2914668/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn an integrated multitrophic aquaculture (IMTA) system, seaweeds serve as extractive species that utilize excess nutrients thereby reducing the risk of eutrophication and promoting sustainable aquaculture. However, the use of excessive fish feeds and the resultant fecal waste as nutrient streams can contribute to variations in nitrogen and phosphorus levels (e.g., primarily NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e) in the surrounding area, and this may impact the physiology of the integrated seaweeds particularly on how these species take up inorganic nutrients. In this study, the effect of different PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e levels on NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake of the three commercially important eucheumatoids \u003cem\u003eKappaphycus alvarezii\u003c/em\u003e, \u003cem\u003eKappaphycus striatus\u003c/em\u003e and \u003cem\u003eEucheuma denticulatum\u003c/em\u003e was examined under laboratory conditions. Seaweed thalli (n = 4) were incubated in seawater media containing 30 µM NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, representing eutrophic conditions, and 0, 0.5, 1.0, 1.5, 3.0 or 5.0 µM\u0026nbsp; \u0026nbsp;PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e for 1 h under a saturating light level of 116 ± 7.13 µmol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e inside a temperature-controlled laboratory. Species-specific responses to PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e levels were observed. For \u003cem\u003eK. alvarezii\u003c/em\u003e, maximum NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake (17.8 ± 1.6 µmol gDW\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e) was observed at 0.5 µM PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e and the uptake rate declined at higher PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e levels. For \u003cem\u003eK. striatus\u003c/em\u003e, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake increases with increasing PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e levels, with maximum N-uptake (6.35 ± 0.9 µmol gDW\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e) observed at 5.0 µM PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e. For \u003cem\u003eE. denticulatum, \u003c/em\u003emaximum NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake (14.6 ± 1.4 µmol gDW\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e) was observed at 1.0 µM PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e. Our results suggest that, among the three eucheumatoid species, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake of \u003cem\u003eK. striatus\u003c/em\u003e persist even at high levels of PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3\u003c/sup\u003e. However, our results also showed that \u003cem\u003eK. striatus\u003c/em\u003e had the lowest range of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake rates. These results should be taken into consideration when incorporating eucheumatoids in IMTA system where PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-3 \u003c/sup\u003elevels significantly vary in space and time.\u003c/p\u003e","manuscriptTitle":"Impacts of aquaculture nutrient sources: ammonium uptake of commercially important eucheumatoids depends on phosphate levels 1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-15 14:21:07","doi":"10.21203/rs.3.rs-2914668/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-07-07T02:10:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-07-01T13:26:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63a81be7-9f07-482b-992c-bc06d6f2f33d","date":"2023-05-19T12:28:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-05-16T12:14:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-05-11T11:25:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-05-11T07:16:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Phycology","date":"2023-05-10T06:37:22+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":"f5d4ee2b-3032-4f3e-a42f-5f9a05a169ae","owner":[],"postedDate":"May 15th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-17T10:22:51+00:00","versionOfRecord":{"articleIdentity":"rs-2914668","link":"https://doi.org/10.1007/s10811-023-03073-1","journal":{"identity":"journal-of-applied-phycology","isVorOnly":false,"title":"Journal of Applied Phycology"},"publishedOn":"2023-09-14 10:22:51","publishedOnDateReadable":"September 14th, 2023"},"versionCreatedAt":"2023-05-15 14:21:07","video":"","vorDoi":"10.1007/s10811-023-03073-1","vorDoiUrl":"https://doi.org/10.1007/s10811-023-03073-1","workflowStages":[]},"version":"v1","identity":"rs-2914668","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2914668","identity":"rs-2914668","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}