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

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 �sh 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-specic 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 signi�cantly vary in space and time.


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 signi cant 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 e uents ( The results of our study have important implications on selecting which species are to be incorporated in an IMTA.

Materials and Methods
Collection, identi cation, 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 identi ed using DNA barcoding (Roleda et al. 2021).The collected samples were acclimated, given su cient time to heal wounds, and then starved overnight in a nutrient-deplete arti cial 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 lled 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 ltered using a 200 µm polyester lter bag, and seawater samples were obtained to measure the inorganic nutrient concentrations spectrophotometrically.All nutrients were found to have undetectable concentrations.
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 ask 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: 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, signi cant 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 signi cant variations were observed.
The ANOVA was performed after the normality (Shapiro-Wilk test) and homoscedasticity (Levine's test) of data were satis ed.All signi cance 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 signi cantly 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.

Discussion
The three eucheumatoid species examined in our study were able to signi cantly reduce NH 4 The NH 4 + uptake rates of eucheumatoids under different PO 4 − 3 levels were species-speci c, 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 andRamus 1984).This is also supported by several published 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 su cient 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, lling 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 e ciency (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 .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 e ciency 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. alvarezii 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 re ect 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 signi cant 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 veri ed 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 su cient 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.

+
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 e ciency of eucheumatoids in absorbing NH 4 + ions.Furthermore, K. alvarezii and E. denticulatum had also been shown to be e cient in absorbing other nutrients like PO 4 − 3 , NO 3 − and NO 2 − (Hayashi et al. 2008; Kambey et al. 2020).These ndings, along with those of our current study, indicate that the use of eucheumatoids as bio lters in an IMTA can successfully reduce the detrimental effects of eutrophication.

BCV Narvarte :FiguresFigure 1 2 Mean
Figures (Buschmann et al. 1996;Neori et al. 2004g 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).
(Ferrera et al. 201601) of seaweeds as bio lters in an IMTA are dependent on several culture conditions(Buschmann et al. 2001).For instance, seaweeds in an IMTA system are subject to uctuating 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 sh 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.Speci cally, little information is available

Table 1 Range
(Bachmann et al. 1996;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)