Seasonal ammonium uptake kinetics of four brown macroalgae: Implications for use in integrated multi-trophic aquaculture

The combined culture of fed species (bivalves, fish) and macroalgae, known as integrated multi-trophic aquaculture (IMTA), has been suggested as a method of mitigating localised nitrogen (N) increase from aquaculture, whilst simultaneously culturing macroalgae for commercial applications. The development of IMTA requires an understanding of the N ecophysiology of candidate macroalga species. We examined seasonal variations in ammonium (NH4+) uptake kinetics, carbon to nitrogen (C:N) ratio, pigment content and soluble tissue N of four macroalgae of the phylum Ochrophyta, Ecklonia radiata, Macrocystis pyrifera, Lessonia corrugata, and Phyllospora comosa, from Tasmania, Australia. This study aimed to determine, (1) if the N physiology of the four macroalgal species was suitable for IMTA applications and (2) whether the species had seasonal variations in N ecophysiology which would influence their suitability for IMTA. Macrocystis pyrifera, L. corrugata, and E. radiata exhibited saturable NH4+ uptake kinetics, with a maximum uptake rate (Vmax) during spring, summer and autumn of 200, 45.8 and 45 μmol gDW-1 h-1 and half-saturation constants (Ks) of 361.3, 104.2 and 121 μM, respectively. Phyllospora comosa exhibited biphasic uptake patterns for three out of four months sampled. There were no noticeable seasonal patterns in pigment content or soluble tissue N for any species. C:N ratios increased from spring (October) to autumn (March) in both E. radiata (28.34 – 47.83) and P. comosa (24.99 – 51.62), indicating progressive N limitation though summer and into autumn. Results suggest that M. pyrifera and P. comosa are most suitable for IMTA due to their high NH4+ uptake potential.


Introduction
Anthropogenic N inputs into coastal waters have been increasing over the last century (Vitousek 1997;Seitzenger et al. 2002;Seitzinger et al. 2008), in part caused by intensive finfish aquaculture, as fish excrete additional NH 4 + into the ecosystem (Handy and Poxton 1993;Wild-Allen et al. 2010). Such inputs can have broader scale ecological impacts outside of the farm footprint (Oh et al. 2015). Nutrient loading can cause eutrophication and anoxic sediments leading to broader scale changes in marine community structure such as alteration of benthic fauna and native fish abundance, increased macroalgal growth and epiphyte loading (Black 2001;Read and Fernandes 2003;Soto and Norambuena 2004;Buschmann et al. 2006;Cubitt et al. 2008).
Integrated multi-trophic aquaculture (IMTA) is being tested as a mitigation solution for the effects of increased dissolved inorganic nitrogen (DIN) loading associated with intensive mono-specific aquaculture operations (Wu et al. 2015;Biswas et al. 2020;Knowler et al. 2020;Rugiu et al. 2021). In these systems, fed aquaculture species are farmed in conjunction with extractive species such as macroalgae which take up a portion of the excreted nutrients and can reduce the overall nutrient input into the environment (Chopin et al. 2001;Chopin 2006). Other organisms in the water column also take up DIN surrounding aquaculture facilities including phytoplankton, but IMTA operations utilise species which can then be harvested for commercial gain (Knowler et al. 2020). They can also provide key ecosystem services such as oxygenation and mitigate against coastal ocean acidification (Hasselström et al. 2018;Fernández et al. 2018). IMTA principles have been applied to aquaculture operations globally including Korea (Park et al. 2018), China (Wu et al. 2017), Chile (Buschmann et al. 1994, Vásquez 2008, Europe (Haglund and Pedersén 1993;Sanderson et al. 2008), Israel (Ashkenazi et al. 2019), and North America (Chopin et al. 1999;Carmona et al. 2006). Similarly, there is a strong interest to implement IMTA methods into Australian aquaculture facilities using local macroalga species (Kelly 2020).
Nitrogen is an essential nutrient for macroalgae, found in chlorophylls a and b, amino acids and cellular enzymes, thus is a key factor limiting macroalgal growth in the marine environment (Hurd et al. 2014). The two main sources of N used by macroalgae are ammonium (NH 4 + ) and nitrate (NO 3 -). Ambient concentrations of inorganic N are generally low in seawater, ranging between <5-20 μM depending on location, however concentrations of NH 4 + can exceed 150 μM immediately surrounding finfish aquaculture facilities (Neori and Shpigel 1999;Carmona et al. 2006). In temperate systems the availability of DIN varies seasonally being generally higher in winter and lower in summer (Hurd et al. 2014). However, spatial variations in inorganic N supply due to anthropogenic inputs can cause localised increases in N concentration regardless of the season (Herbert 1999;Howarth and Marino 2006;Van Alstyne 2018).
One key aspect to assess macroalgal species suitability for IMTA is understanding NH 4 + uptake kinetics (Roleda and Hurd 2019) because it is the primary waste product of operations incorporating marine finfish, excreted through the gills (Randall and Wright 1987;Wilkie 1997). Also localised increases in NH 4 + concentrations are observed surrounding finfish cages (Sanderson et al. 2008). NH 4 + is readily available to macroalgae, where it is taken up through cell membranes via one or more of three mechanisms -passive diffusion, facilitated diffusion and active uptake (Hurd et al. 2014;Roleda and Hurd 2019). Passive transport involves uptake via simple diffusion, whereas facilitated diffusion and active transport utilise proteins to move ions into the cell.
Uptake kinetics can be used to gain an understanding of the mechanisms of inorganic N uptake (Harrison and Druehl 1982;Rosenberg and Ramus 1984;Phillips and Hurd 2004;Roleda and Hurd 2019). Active uptake is indicated by a plot of uptake rate vs. concentration exhibiting saturating kinetics and can be described by the Michaelis-Menten equation (Hurd et al. 2014): From this relationship, the parameters maximum uptake rate (V max ) and half-saturation constant (K s ) are determined. Desirable V max and K s values for IMTA are dependent on V = V max S K m + S the objective of the IMTA operation (Chopin et al. 2001). Species with a high V max and K s values can better take up NH 4 + at high concentrations, which is desirable for IMTA applications and lower values of K s indicate a greater ability to procure nutrients at a low concentration. Passive uptake is indicated by a linear relationship between concentration and uptake rate. A combination of linear and saturating components indicates that both active and passive uptake mechanisms are present, known as biphasic or multi-phasic uptake (Roleda andHurd 2019, See Fig. 6.2 d in Hurd et al. 2014). Biphasic uptake mechanisms also prove desirable for IMTA applications, as the species can take up NH 4 + at high external NH 4 + concentrations. In addition to NH 4 + uptake kinetics, determining which macroalgal species are most suited to IMTA requires a comprehensive understanding of their underlying N ecophysiology (Neori et al. 2004). Of particular importance are the C:N ratios, photosynthetic pigment content, and soluble tissue nitrogen pools which are used to assess the nitrogen status of the macroalgal tissue (Roleda and Hurd 2019;Rugiu et al. 2021). Comparatively high C:N ratios, low pigment content and low soluble tissue N pools can indicate that macroalgal tissues are depleted in N (Roleda and Hurd 2019;Chopin et al. 1995;Vergara et al. 1993). N depleted species are able to uptake excess DIN in the environment, such as that released from aquaculture operations and are therefore useful in IMTA operations (Pedersen and Borum 1997;Hadley et al. 2018). Soluble tissue N is the amount of N that is stored within the macroalgae cells and provides an indication of nutrient storage capacity, and whether the macroalgae are N depleted at the time of sampling (Roleda and Hurd 2019). Seasonal variation in macroalgal N physiology has been observed for many temperate regions worldwide due to changing light, water temperature, wave motion and nutrient supply (Kain 1989;Lüning 1993). Macroalgae adapt to these changes by seasonally altering pigment content (Flukes et al. 2015), nutrient uptake and storage (Asare and Harlin 1983, Hurd and Dring 1990, Phillips and Hurd 2004, as well as biochemical composition (Wheeler and Björnsater 1992).
Macroalgae of the orders Laminariales and Fucales are key components of temperate reefs worldwide and are candidate species for IMTA in southern Australia. In Tasmania, there are a range of animal aquaculture operations including salmon, mussels, abalone and pacific oysters (DPIPWE 2020), with growing interest to develop IMTA to help mitigate DIN inputs and provide a commercial product. The proportion of DIN up taken my macroalgae in an IMTA setting varies depending on location, stocking densities and hydrodynamics of the area. Modelling studies in Tasmania indicate that M. pyrifera can remove up to 11% of DIN input from salmon aquaculture over a nine-month period (Hadley et al. 2018). Four macroalgal species, Ecklonia radiata (C. Agardh) J. Agardh, Macrocystis pyrifera (Linnaeus) C.
Agardh, Lessonia corrugata A.H.S. Lucas, and Phyllospora comosa (Labillardière) C. Agardh, have been identified as potential species for IMTA operations in Tasmania due to economic value and potential for high biomass production (Sanderson and Di Bendetto 1988;Kelly 2020). Members of the Laminariales are being trialled in other regions due their comparatively fast growth rate (Barrington et al. 2009), and although the growth rates of the Tasmanian kelps are not well studied, Macrocystis and Ecklonia are known to have growth rates similar to those of other Laminariales (Miller et al. 2011;Schiel and Foster 2015). Here we determined (1) if the NH 4 + physiology of the four Tasmanian species studied were suitable for IMTA applications and (2) if the species exhibited seasonal patterns in their NH 4 + physiology that affect their suitability for IMTA.

Sample Collection
Macroalgal samples were collected sub-tidally at 3-5 m depth from two sites in southern Tasmania. Collection occurred four times during Spring -Autumn 2018-2019 at Flowerpot Point, Blackmans Bay (-43° 0'27"S, 147°19'44"E) and the Tessellated Pavements, Eagle Hawk Neck (43° 0'30"S, 147°56'7"E) ( Table 1). Mature blades were collected for M. pyrifera and L. corrugata. Mature, lateral blades were collected for E. radiata and P. comosa. Five individuals for each species (n = 5) were collected at each sampling event. Samples were individually wrapped in damp tissue paper, stored in a dark cool-box and transported back to the laboratory, with 30 min for Flowerpot Point and 60 min for Tessellated Pavements. At the laboratory, samples were immediately wiped with tissue to remove any epibionts and rinsed in filtered, UV sterilised seawater (filtered to 1 μM and UV-sterilised with an Emperor Aquatics Smart HO UV steriliser, 025050-2, 50 W lamp) before being divided into experimental sections (see below). At the time of each collection, replicate 10 mL water samples (n = 3) were taken for analysis of ambient NH 4 + and NO 3 in seawater. Samples were filtered through a 0.7 μm filter (Whatman GF/F) on-site before being transported back to the laboratory and frozen at -20°C until analysis.

NH 4 + Uptake Kinetics
To determine the NH 4 + uptake kinetics of each species, individual blades from each species (n = 5) were divided into seven discs using a 3 cm diameter cork borer for M. pyrifera (~ 0.5 g), E. radiata (~0.5 g) and L. corrugata (~ 1.0 g), or seven 5 cm individual apical blade sections for P. comosa (~0.25 g). Samples from each individual were placed into separate beakers with filtered seawater and placed on shaker tables set to 100 rpm and a photoperiod of 12:12, which was kept constant across each experiment (at 150 μmol photons -1 m -2 s -1 ) for 24 h to allow wound healing (McDowell et al. 2015).
To determine the maximum uptake rate (V max ) and halfsaturation coefficient (K s ) of each macroalga species, a multiple flask, constant incubation time, experiment was conducted for (Philips and Hurd 2003) at each sampling event, within 48 h of sample collection. A total of four experiments were conducted for each species. For each species, 37 × 250 mL conical flasks were filled with 200 mL of filtered seawater and enriched with NH 4 + from a stock solution of NH 4 Cl (0.2 M) to give a concentration series of approximately 2, 10, 20, 40, 80, 160 and 240 μM, with five replicates (n = 5) for each species. Two additional conical flasks with no macroalgae were used as controls, with one containing filtered seawater and one with filtered seawater enriched to 240 μM.
Before addition of the macroalgae, an initial seawater sample was taken from each flask with a 12 mL syringe filtered through a 0.7 μM filter (Whatman GF/F). Samples were stored in 12 mL polyethylene tubes at -20°C. One piece of alga was then placed into each flask and set on a shaker table at 100 rpm under 150 μmol photons m -2 s -1 . All flasks were left for two h as no lag or surge phases were detected for any species in the preliminary time-course experiment (data not shown). After two hours, a final water sample was taken, and macroalgae were removed from each flask. Macroalgae were blotted dry, weighed for wet weight (WW) and photographed for surface area. The surface area was calculated using Adobe Photoshop CC 2018 (Adobe Software

Seawater nutrient analysis
NH 4 + concentrations from the uptake experiments and NO 3 and NH 4 + concentrations extracted from tissue (soluble pools) was determined using a QuickChem 8000 Automated Ion Analyser (LaChat Instruments) using the methods outlined in 'Determination of nitrate/nitrite in brackish or seawater by flow injection analysis' (Diamond 2008) and 'Determination of ammonia in brackish or seawater by flow injection analysis' (Liao 2008).

Calculation of NH 4 + uptake rates
Uptake rates of NH 4 + in individual flasks were calculated using the following equation: where V = uptake rate (μmol g -1 DW h -1 ), S i = initial concentration of seawater NH 4 + (μM), S f = final concentration after time interval (μM), t = time interval (2 h) and DW = dry weight of macroalgal samples (g) (Harrison and Druehl 1982).

Soluble tissue NO 3 and NH 4 + pools
Soluble tissue NO 3 and NH 4 + pools were determined in November 2018, January and March 2019 by boiling water extraction (Hurd et al. 1996). One additional tissue sample was taken from each replicate (n = 5) of each of the four species prior to the NH 4 + uptake experiment for analysis of soluble tissue N. These tissue samples were blotted dry and cut into sections of 0.25 g ± 0.01 g. Each piece was placed into a 50 mL boiling tube with 20 mL of deionised water and samples were refrigerated overnight (4°C). Test tubes were then placed in a boiling water bath for 20 min. The samples were left to cool, and the liquid was decanted and filtered through 0.7 μm glass filter paper (Whatman GF/F). The extract was stored at -20°C before analysis using an Automated Ion Analyser described above. This process was repeated three times to ensure all soluble tissue N was extracted. NH 4 + and NO 3 contents were calculated using the following equation: where N T is the total concentration of NH 4 + or NO 3 extracted, N 1, N 2 , and N 3 are the concentrations of NH 4 + or NO 3 is the solution after subsequent boiling extractions , V is the volume of water in the boiling tube (L) and WW is the wet weight (g) of the seaweed sample.

Photosynthetic Pigment Content
Photosynthetic pigment content (chlorophyll a, chlorophyll c and fucoxanthin) was determined using the methods outlined in Seely et al. (1972). Samples of 0.1~ 0.15 g wet weight were taken from the five collected replicates of each species, frozen in liquid nitrogen and stored at -80°C until extraction. Samples were then defrosted and placed into test tubes. Although approximately the same weight (0.1-0.15 g) as other species, the blades of L. corrugata were thicker than other species, and so were cut into smaller pieces to facilitate extraction. 4 mL of dimethyl sulfoxide (DMSO) was added to each test tube and samples were left to extract for 10 min. The liquid was then decanted and collected in a test tube. Immediately afterwards 6 mL of 90% acetone v/v was added to the macroalgal tissue and left to extract for 30 min or until tissue was void of pigments, and subsequently decanted into a separate test tube. Test tubes were kept over ice and regularly agitated. Absorbance of the extracts were measured with a S-22 UV/Vis Spectrophotometer (Halo RB-10, Dynamica Scientific Ltd). The absorbance of the DMSO extract was measured at 665, 631, 582, and 480 nm and the acetone extract measured at 664, 631, 581 and 470 nm. Pigment contents were calculated using the equations given by Seely et al. (1972).

C, N and C: N ratio
Tissue carbon, tissue nitrogen and C:N ratio of three replicates per species were determined in October and November 2018 and January and March 2019 using the methods described in Cornwall et al. (2015). An additional 2 cm diameter disk was taken from each blade of the collected species and dried at 60°C for 48 h. A Carlo-Erba NA1500 elemental analyser coupled to a Thermo Scientific Delta V Plus via a Conflo IV was used in analysis with combustion and reduction of samples was achieved at 1020°C and 650°C, respectively. Values were normalised to the Vienna Pee Dee Belemnite (VPDB) scale with a 3-point calibration and both precision and accuracy were ± 0.1 % (1 SD).

Curve fitting and data analysis
The Michaelis-Menten function (i.e., a rectangular hyperbola) was fitted to each replicate of M. pyrifera, L. corrugata, and E. radiata using SigmaPlot (Systat Software Inc), and V max and K s obtained for each replicate. The mean values for V max and K s ± SE were then obtained for each species in each sampling month.
For P. comosa the Michaelis-Menten function could only be fitted for data collected in January. In October, December, and March, the pattern of uptake vs. concentration was biphasic with uptake of NH 4 + appearing to saturate for concentrations <160 μM, however further increased linearly between 160 -240 μM. Michaelis-Menten curves were therefore fitted to uptake rates at NH 4 + concentrations <160 μM, and a linear regression was applied to uptake rates at NH 4 + >160 μM. Negative uptake values were excluded from curve fitting and manuscript figures but are presented in Appendix 1.
The means ± SE were calculated for soluble tissue NH 4 + and NO 3 -, pigment content, C:N ratio, C and N. Data were analysed using the statistical software R (R core development team 2017). Data were tested for conformity of assumptions of homogeneity of variances and normality of residuals by plotting residuals and fitted values. Transformations to meet these assumptions for V max , K s , soluble tissue N and pigment determined using the Box-Cox method, and log transformations were applied to the data before analysis. A two-way ANOVA with the factors 'Species', 'Season' and 'Species*Season' was applied where p < 0.05. This test compared differences between species, between season and interaction effects and was applied to V max , K s , soluble tissue N content, pigment content, and C:N ratio. For significant results, a-posteriori multiple comparisons were then conducted using a Tukey's HSD test to elucidate specific differences between species across seasons. Additionally, Pearson's correlation test (p < 0.05) was used to determine the relationship between V max and K s across the sampling months.

Background seawater nutrient concentration
Ambient, in situ seawater nutrients did not exceed 3.32 μM, with the highest concentrations recorded at the Tessellated Pavements ( Table 2). The range of NO 3 concentrations across seasons were similar at both Flowerpot Point and the Tessellated Pavements.

NH 4 + uptake kinetics
Controls showed minimal change in NH 4 + concentration (0 -10 %) over the two-hour experimental period and the depletion of NH 4 + from the seawater with macroalgae was thus attributed to macroalgal uptake. L. corrugata, E. radiata, and M. pyrifera all exhibited saturable uptake (Fig. 1). In contrast, P. comosa uptake was biphasic for three of four sampling events (Fig. 2) and V max and K s values could not be determined.
V max were significantly different between species (p < 0.05), and an interaction effect between species and season (p < 0.05), indicating that seasonal patterns in V max were not consistent across species (Table 3, Fig 3). An a-posteriori Tukey's HSD test revealed no significant differences in E. radiata and L. corrugata V max over the sampling months (p > 0.05). In contrast, M. pyrifera V max values varied seasonally and were highest in October 2018 (159 μmoles g -1 DW h -1 ± 23 SE, Tukey's test, p < 0.01) and January 2019 (200 μmoles g -1 DW h -1 ± 23 SE, Tukey's Test, p < 0.01) ( Table 3). The highest V max values for M. pyrifera were approximately 4.5 times greater than those for E. radiata and L. corrugata, which ranged between 17 and 45 μmol g -1 DW h -1 .
For K s , the two-way ANOVA indicated significant differences in species and season (Table 3). As with V max , an interaction effect of species and season was also present for K s values, indicating that seasonal effects were not consistent across species. Graphs of mean K s ± SE are shown in Fig. 3. The K s of L. corrugata did not change significantly between sampling months (Tukey's test, p > 0.05), ranging between 104 and 134 μM. In contrast, the K s of E. radiata varied over the sampling months (Tukey's test, p < 0.05) and was approximately nine times higher in March 2019 (204.33 μM ± 52.35 SE) than October 2018 (23.50 μM ± 5.05 SE). The K s of M. pyrifera also varied across sampling months, with November K s values being significantly lower than the other seasons (Tukey's test, p < 0.05). Higher K s values were positively correlated to higher V max values (Pearson correlation, R = 0.808, t = 10.45, df = 58, p < 0.01

Soluble tissue NO 3 and NH 4 + pools
Soluble tissue NO 3 and NH 4 + content (μmol g -1 WW) were variable both across seasons and between species (Table 3). Additionally, an interaction effect of species and season was present with no clear seasonal pattern in soluble tissue N content. For all species, NH 4 + content was higher than NO 3 content for all seasons (Fig. 4). As such, variation in total N content reflected changes in tissue NH 4 + . Average NH 4 + For L. corrugata, total soluble N content was lowest in summer (January) (0.928 μmol g -1 WW ± 0.140 SE, Tukey's test p < 0.01) when compared to November and March (~4 Rates of NH 4 + uptake as a function of NH 4 + concentration for P. comosa. Note the y-axis scale range. Each point is a replicate seaweed (n = 5) μmol g -1 WW). A similar pattern was seen in M. pyrifera, where total soluble N content was approximately three times higher in March (5.435 μmol g -1 WW 1 ± 1.090 SE, Tukey's Test p < 0.01) than January (0.581 μmol g -1 WW ± 0.114 SE). In contrast, total soluble N content for E. radiata in January was approximately double the values recorded for November and March (4.519 μmol g -1 WW ± 0.693 SE, Tukey's test p < 0.01). P. comosa soluble tissue N content was highest in November (2.42 μmol g -1 WW ± 0.292 SE, Tukey's test, p < 0.01).

Photosynthetic pigment content
Pigment content was significantly different between species and between seasons (Table 3). An interaction effect of species and season was also present (p < 0.05). Lowest mean pigment contents were recorded for L. corrugata samples in January 2019 (0.0591 mg g -1 ± 0.0139 SE) and were highest in P. comosa in November 2018 (0.326 mg g -1 ± 0.138 SE) (Fig. 5). There were no significant differences in pigment content across season for L. corrugata, M. pyrifera and P. comosa (Tukey's test, p > 0.05 for all pairwise comparisons). Mean total pigment content of E. radiata was approximately three times higher in October (0.309 mg g -1 ± 0.0212 SE) than January and March (p < 0.01). November pigment content was lower than October (0.149 mg g -1 ± 0.0167 SE, p > 0.01) and the lowest mean total pigment content for this species was recorded in March (0.0898 mg g -1 ± 0.0119 SE).

C, N and C:N ratio
The two-way ANOVA revealed differences in tissue N, tissue C and C:N ratio between seasons and species, with and additional interaction effect (Table 3). For all species,  comosa. Bars represent mean ± SE, n = 5. * denotes significant difference in total pigment content from other months within a species (Tukey's test,p < 0.05) tissue nitrogen did not exceed 1.4%. For E. radiata and P. comosa, tissue nitrogen decreased by approximately half from October to March (p < 0.01) (Fig. 6). Highest N values were recorded in November for L. corrugata (1.38% ± 0.017 SE) and January for M. pyrifera (1.06% ± 0.012 SE). The highest tissue C values were in E. radiata and P comosa samples, where C content was always above 30%. For L. corrugata, tissue C increased between October 2018 and March 2019 (Tukey's test, p < 0.01). For M. pyrifera, tissue C was highest in November and January, at 27.6% and 28.72% respectively. The C:N ratio for E. radiata increased between October 2018 and March 2019 (Tukey's test, p < 0.05 for all pairwise comparisons). The same trend was seen in P. comosa. L. corrugata and M. pyrifera showed no clear seasonal pattern in C: N ratio across the sampling months.

Discussion
For IMTA applications with a primary goal of reducing NH 4 + output into the environment, macroalgal species with a high potential for DIN uptake (high V max ) are required. The maximum V max was recorded for M. pyrifera and was ~ 4.5 times greater than for L. corrugata and E. radiata. The higher nutrient uptake rates seen for M. pyrifera are consistent with the fast growth rate of this species (Gerard 1982) and higher NH 4 + uptake rates have been observed in species with high growth rates (Pedersen and Borum 1997). M. pyrifera exhibited the highest K s value, indicating that this species is better able to take up NH 4 + at higher concentrations, compared to species with a lower K s (Roleda and Hurd 2019). Concentrations of NH 4 + immediately surrounding fin fish aquaculture facilities can exceed 150 μM (Neori and Shpigel 1999;Carmona et al. 2006), compared to background concentrations of DIN of < 20 μM, which as suggested by our results would be rapidly taken up by M. pyrifera. Indeed, it has been utilised in IMTA in Chile (Buschmann et al. 2008) and has a high market value compared to other kelp species (Correa et al. 2016;Camus et al. 2019). Of the four species studied here, M. pyrifera appears the most suitable for IMTA operations in Tasmania.
M. pyrifera, L. corrugata and E. radiata all exhibited saturable NH 4 + uptake kinetics. Saturable NH 4 + uptake is common in macroalgae, and a summary of species exhibiting saturable NH 4 + uptake is provided in Table 3. Active uptake mechanisms are particularly important is regions where concentrations are low and can be limiting, as algae must be able to actively pump DIN into their cells against a concentration gradient, for storage and growth. In contrast to the laminarians, the fucalean brown seaweed P. comosa exhibited biphasic uptake patterns for all months except summer (January). To our knowledge, this is only the third record of biphasic NH 4 + uptake in the class Phaeophyceae, and biphasic uptake has been observed only for members of the Fucales: Fucus distichus (Thomas et al. 1985) and Fucus spiralis (Topinka 1978).
The likely presence of both active and passive N uptake mechanisms, as indicated by bi-phasic uptake as seen in P. comosa, is considered an adaptation to areas with large variations in nutrient concentrations (Collos et al. 1997;Lomas and Glibert 1999). Biphasic uptake allows the macroalgae to operate active uptake when nutrient concentrations are low, and passive uptake when nutrient concentrations are high (Buchanan et al. 2000). As such, P. comosa may have uses in IMTA operations as its biphasic uptake mechanisms Fig. 6 Tissue C (A) and tissue N (B) expressed as % of total DW and C:N ratio (based on %) across sampling months. Bars represent mean ± SE, n=3. * denotes significant difference (Tukey's test, p < 0.05) from other sampling months of the same species  To compare the values of V max and K s obtained in our study with those of other macroalgal species, a search of published literature was undertaken and the uptake kinetics of 62 species are reported in Table 4. There were 26 Rhodophyta, 24 Ochrophyta and 16 Chlorophyta, in addition to the four Ochrophyta species studied here. In general, the phyla Chlorophyta and Rhodophyta had higher V max and K s values than the phylum Ochrophyta, Class Phaeophyceae. This is the first study to examine the NH 4 + uptake mechanisms of P. comosa and L. corrugata, and the V max for these species fell within the range of studies on macroalgae of the order Laminariales. For E. radiata, Paling (1991) suggested a passive uptake mechanism for NH 4 + where uptake rate increased proportionally to NH 4 + concentration, but we found evidence of active uptake as saturating kinetics were observed.
For kelps, a C:N ratio of >15 -20 is often considered to indicate N limitation (Hurd et al. 2014). For some macroalgae from mid and low latitudes, there are strong seasonal patterns of tissue nitrogen and C:N ratio. For example, C:N ratios of Ulva olivascens have been shown to change from 12.9 to 39.4 moving from spring to summer (Altamirano et al. 2000) due to higher levels of nitrogen in the water column in winter months. Such seasonal patterns in soluble tissue N content, N uptake, pigment content and macroalgal growth have been demonstrated by Topinka (1978), Küppers and Weidner (1980), Asare and Harlin (1983), Rosell and Srivastava (1985), Brown et al. (1997), Abreu et al. (2011) and Bearham et al. (2013). In this study, tissue N in E. radiata and P. comosa declined over summer and C:N ratio increased, indicating progressive N limitation from spring to summer. In contrast, L. corrugata and M. pyrifera showed no clear seasonal pattern in C:N ratio. The C:N ratio of M. pyrifera, E. radiata, and P. comosa was >20 for all months, indicating these species are N limited year-round (Hurd et al. 2014) and would therefore be able to uptake NH 4 + all year in an IMTA scenario. Lessonia corrugata was the only species which exhibited a relatively low (<20) C:N ratio year-round. As seawater DIN at the collection locations remained relatively constant over the sampling months, it is possible that E. radiata and P. comosa had increased growth in the summer months due to higher irradiance and warmer waters, and therefore used any stored N. Additional DIN sources from IMTA operations may prove beneficial for the growth of the species during summer.
Of the species studied, M. pyrifera and P. comosa appear to be the most suitable for IMTA applications in Tasmanian waters due to their high NH 4 + uptake potential. Lessnia corrugata and M. pyrifera did not show any distinct patterns in N ecophysiology that could be attributed to the changing seasons, however P. comosa had a depletion in tissue N over the summer, indicating N limitation. Other studies have proposed the introduction of a multi-cultured approach at which different species are grown at different depths. Experiments conducted in Chile have demonstrated that M. pyrifera can be best cultivated at 3 m and can be farmed in conjunction with Gracilaria chiliensis (Buschmann et al. 2008). This approach may be an option in Tasmania. Ecklonia radiata and L. corrugata have high commercial value as food products (Sanderson and Di Bendetto 1988) and as sources of extracts (Lorbeer et al. 2015). Farmed in conjunction with a species of high environmental value, such as M. pyrifera, a multi-cultured approach may be the best option environmentally and economically.
As the commercial markets for farmed seaweeds grow, the methods used in this study may be used to assess the suitability of other local species including Rhodophyta and Chlorophyta for use in IMTA operations. The work conducted in this study is the first to confirm that M. pyrifera in Tasmania has desirable NH 4 + uptake kinetics for use in IMTA. Additionally in the exploration of NH 4 + uptake kinetics of P. comosa, the works identify a potential species for IMTA that has, to the best of our knowledge, not previously been utilised in aquaculture operations.

Supplementary Information
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