Changes in metabolism, growth and nutrient uptake of Ulva fasciata (Chlorophyta) in response to nitrogen source

Algal Research 46 (2020) 101781

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Changes in metabolism, growth and nutrient uptake of Ulva fasciata (Chlorophyta) in response to nitrogen source

T

Ben Shahara,b, Muki Shpigelc, Roy Barkana,b, Matan Masasaa,b, Amir Neoric,d, Helena Chernova, ⁎ Eitan Salomona, Moshe Kiflawib,d, Lior Guttmana, a

National Center for Mariculture, Israel Oceanographic and Limnological Research Institute, Eilat 88112, Israel Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel c Morris Kahn Marine Research Station, The Leon H. Charney School of Marine Sciences, University of Haifa, Haifa 3498838, Israel d The Interuniversity Institute for Marine Sciences of Eilat, Eilat 88103, Israel b

A R T I C LE I N FO

A B S T R A C T

Keywords: Aquaculture Biofiltration Metabolism Nitrogen Phosphorus Ulva fasciata

The form of inorganic nitrogen (N) determines the biofiltration performance of Ulva, particularly by inhibition of nitrate assimilation in the presence of ammonia. In the current study, Ulva fasciata from a biofilter of fishpond effluent was examined for its biomass production, photosynthetic activity, nutrient uptake and activity of nitrate reductase when supplied with either ammonia or nitrate as the sole nitrogen source. The effects of prior acclimation to either nitrogen source were also studied. Cultivation of Ulva with ammonia resulted in a rapid uptake of this nutrient, whereas photosynthesis, uptake of orthophosphate and C:N ratio were greater in cultures with nitrate. The transfer of Ulva from ammonia to nitrate nutrition increased carbohydrate content in the biomass. Longer culture in nitrate did not influence photosynthetic yields, growth, or N and P uptake rate by the algae, but increased both lipid and P content in biomass. Once transferred from ammonia-rich to nitrate-rich seawater, nitrate uptake by Ulva commenced immediately, although the activity of the enzyme nitrate reductase was first detected only 28 h later. Culture of Ulva in different nitrogen forms, ammonia and nitrate, may determine application of the resulting biomass in different industries, e.g., feeding, bioethanol, biofiltration, etc., considering the rapid production of protein-rich Ulva in the presence of ammonia, while yield is lower in the presence of nitrate but the biomass contains more carbohydrates and removes P from the effluent more rapidly.

1. Introduction Accumulation of nitrogen and phosphorus excreted by fish in intensive mariculture effluents is a major concern in the fish farming industry [1]. In land-based integrated multi-trophic aquaculture (IMTA) systems, algae, particularly Ulva spp., efficiently utilize N from fishpond effluents [2–5]. Yield of Ulva in IMTA systems may reach ~55 g dry weight (DW) m−2 d−1 [6], among the highest values reported for plants [7]. Moreover, protein content in effluent-grown Ulva is high, up to 37% of dry biomass [8], making IMTA-grown Ulva a nutritious feed for the culture of various aquatic organisms, including abalone [9], sea urchins [10,11] and fish [12]. While ammonia (as NH3N + NH4-N, here referred to as total ammonia nitrogen, TAN) is the primary N waste in fish excretion [13,14], its subsequent oxidation in culture tanks and pipes by chemoautotrophic nitrifying bacteria leads to the accumulation of nitrate (as NO3-N) [15]. The uptake and assimilation of TAN and NO3-N require different

⁎

strategies by the algae. NH4 uptake is conducted through either low- or high-affinity transporters (i.e., according to substrate concentration) while at high pH, un-ionized ammonia diffuses through the cytoplasmic membrane into the cells [16]. Once inside the cytoplasm, Ulva metabolizes TAN directly to amino acids through the glutamine synthetase glutamine:2-oxoglutarate amidotransferase (GS-GOGAT) pathway. In comparison to TAN, NO3-N assimilation is slower. It requires high energetic input, since the active transport of NO3-N into the cells is mandatory [17], and is followed with subsequent reduction events by the endogenic enzymes nitrate reductase (NR) and nitrite reductase (NIR) to form NH4+, which can be further integrated through the GSGOGAT pathway [18]. Moreover, even low levels of TAN inhibit NO3-N uptake by Ulva [19–22] via the inactivation of NR [23–25]. Since assimilation of nitrogen requires carbon skeletons for amino acid synthesis, the uptake rate of N is coupled with C metabolism [26], which in turn depends on P in several incorporation and transformation pathways [27]. Moreover, in their study, Huppe et al. [28] indicated

Corresponding author. E-mail address: [email protected] (L. Guttman).

https://doi.org/10.1016/j.algal.2019.101781 Received 25 July 2019; Received in revised form 6 December 2019; Accepted 25 December 2019 2211-9264/ © 2019 Elsevier B.V. All rights reserved.

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of approximately 0.6 mol N m−2 d−1 by exchanging water at a fixed rate of three tank volumes d−1 (Table 1), according to [4,32]. Under such conditions, algae were provided with an excess of nutrients that also allowed a reliable measurement of N uptake [21,22]. Over the four days of the experiment, the abiotic parameters (water temperature, light, pH values), as well as the activity rate of NR, were measured every 4 h between 5:00 AM and 9:00 PM.

different carbon utilization pathways under different N regimes, with activation of the phosphogluconate pathway (the oxidative pentose phosphate pathway) during photosynthetic assimilation of NO3− but not NH4+ by the unicellular green algae Selenastrum minutum. NO3-N metabolism inhibition by TAN in Ulva is a common occurrence, with real-world implications on algal biofiltration. A practical solution to this issue is a two-step Ulva biofilter [19] for the treatment of fishpond effluents, with efficient removal of TAN in the upstream unit followed by efficient NO3-N removal in a downstream nutrients-polishing unit. However, both algae production and N uptake in the downstream Ulva biofilter can be significantly slower than in the upstream biofilter [19]. The current study aim the factors that determine Ulva NO3-N removal by examination of the seaweed's performance (nutrient uptake, photosynthesis, enzymatic metabolism, growth and biochemical composition) when cultured in the presence of either TAN or NO3-N as its sole nitrogen source. The effects of pre-acclimation of Ulva in seawater to either of these nutrients were also examined.

2.2. Biomass production At the end of the experiment, Ulva was harvested from each tank, rinsed in fresh water, drip-dried for 2 h and then measured for fresh weight. Portions of the air-dried biomass (~1.5 g) were analyzed for dry weight (DW) and ash-free dry weight (AFDW), following APHA [33]. The daily growth rate (DGR) was calculated following the recommended equation [34]:

DGR (as%d−1) = 100 × [(Wt /W0)1/t − 1]

2. Materials and methods

where W0 and Wt are the initial and final dry weight of biomass (g), respectively, and t is the number of culture days.

2.1. Ulva culture in various nutrient regimes

2.3. Chlorophyll a fluorescence measurement

Fresh thalli of Ulva were collected from the IMTA facility at the National Center for Mariculture [29] and were acclimated for three weeks in tanks (1.1 × 0.9 × 0.55 m) with nutrient-enriched seawater by either TAN (as NH4Cl) or NO3-N (as KNO3), to set an N:P molar ratio of approximately 10:1 [30] at an initial stocking density of 1 kg fresh weight (FW) m−2. After acclimation, the seaweed was transferred into experimental tanks (0.7 × 0.5 × 0.3 m) at an initial stocking density of 1 kg FW m−2 for four more days. All tanks were positioned outdoors under direct sunlight (952 μmol photons m−2 s−1 ± 62 S.E at 1:00 PM). Bottom-aeration was provided continuously to the tanks [3] to stir the seaweed vertically in the water column and break down diffusive boundary layers at the surface of the thalli which may hinder nutrient uptake [31]. The different nutrient regimes (in three replicates each) included the transfer of Ulva that was cultured in TAN-enriched seawater to tanks with either the same culture conditions (i.e., TAN-enriched seawater, here referred to as TANTAN Ulva) or to tanks with NO3-N-enriched seawater (here referred to as TAN-NO3 Ulva). A third treatment (also in three replicates) included the pre-acclimation of Ulva in NO3-N-enriched seawater and its transfer to the same culture conditions (i.e., NO3-N-enriched seawater, here referred to as NO3-NO3 Ulva) to examine the effects of pre-acclimation in this N source. During the whole cultivation period (i.e., before and after algae transfer to new media), N was supplied to the cultures at an areal load

The photosynthetic quantum yield of Ulva was measured using pulse amplitude modulation fluorometry (PAM) [35]. Measurements were taken upon re-inoculation and 48 h afterward (days 0 and 2 at 12:00 PM), using an Imaging PAM M-Series fluorometer (Walz GmbH, Germany) [36]. Two ~4 cm2 thalli samples from each tank were placed, each in its own well, in 24-well plates. Each well contained 0.4 ml of the respective growth medium. The plates were kept in the dark for 30 min before PAM analysis. Zero fluorescence (F0) was determined for the dark-adapted samples, followed by a saturation light pulse which was set at 3000 μmol m−2 s−1 and lasted 800 ms to obtain maximum fluorescence (Fm). The maximum quantum yield of photosystem II (Fv/Fm) was calculated following the recommended equation [35]:

Fv /Fm = (Fm − F0)/Fm. 2.4. Nutrient analyses Water samples from the inlet and outlet of each tank were filtered (0.45 μm, Whatman GF/C) once a day at 3 PM and analyzed in a segmented flow auto-analyzer (Skalar, the Netherlands), following the manufacturer's recommended methods for TAN [37], NO2-N and NO3-N [38], and PO4-P [39] determination in seawater. The nutrient uptake rate was calculated following the equation:

Table 1 Water flow and nutrients levels in cultures of Ulva fasciata at different nutrient regimes: TAN-enriched seawater (TAN-TAN), transfer from TAN- to NO3-N-enriched seawater (TAN-NO3), or pre-acclimation in NO3-N-enriched seawater (NO3-NO3). Values are mean ( ± SD), n = 3. Regime

Day

Water flux L d−1

N μmol L−1 In

TAN-TAN

NO3-NO3

TAN-NO3

0 1 2 3 0 1 2 3 0 1 2 3

326 276 305 348 301 255 290 292 315 267 305 367

± ± ± ± ± ± ± ± ± ± ± ±

9 15 50 24 12 23 9 22 38 32 50 47

795 818 813 833 679 702 680 677 670 695 664 644

P μmol L−1 Out

± ± ± ± ± ± ± ± ± ± ± ±

124 137 145 108 9 1 7 8 53 33 54 11

2

218 424 309 372 416 641 627 652 484 676 623 600

In ± ± ± ± ± ± ± ± ± ± ± ±

36 150 49 105 26 14 7 5 45 43 79 5

62 70 65 67 74 78 68 70 74 81 73 72

Out ± ± ± ± ± ± ± ± ± ± ± ±

4 7 7 1 5 1 3 1 16 17 15 1

39 59 54 58 24 21 29 24 24 26 31 32

± ± ± ± ± ± ± ± ± ± ± ±

2 10 11 11 2 1 2 2 2 3 2 8

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NUR = (Cin − Cout ) × FR. where NUR is the nutrient uptake rate (μmoles h−1), Cin and Cout are the nutrient concentration (μmoles L−1) in the inlet and outlet of the tank, respectively, and FR is the seawater flow rate (L h−1). The specific uptake rate of each nutrient was further calculated by dividing NUR by the biomass DW in the tank. 2.5. Nitrate reductase (NR) activity During each day of the experiment, NR activity was determined at four-hour intervals over 16 h, starting 1 h before sunrise (i.e., from 5:00 AM to 9:00 PM). Fresh Ulva thalli submerged in their ambient seawater were collected from the culture tanks and transferred directly to the lab for measurement of NR activity by a recommended in vivo assay [8,40]. The thalli were blotted dry between paper towels and the tissue samples were diced and incubated for 60 min in the dark (at 30 °C) in test tubes, each filled with 5 ml of the assay medium which contained 72.2% 1 M dipotassium phosphate, 16.7% 1 M monopotassium phosphate, 10% 1 M potassium nitrate, 1% isopropanol and 0.1% polysorbate 20. The NO2-N concentration in the medium was measured upon initiation and termination of incubation (t0 and t60, respectively) following [41]. The NR activity was calculated following the equation:

Fig. 1. Temperature and light intensity in the cultivation tanks of Ulva fasciata under the different nutrient regimes. The temperature was measured in TANenriched seawater (TAN-TAN, ●), transfer from TAN- to NO3-N-enriched seawater (TAN-NO3, ), or pre-acclimation in NO3-N-enriched seawater (NO3-NO3, ●). Light intensity (*) was measured randomly at the tank surface in the experiment site. Values are mean ( ± SD), n = 3.

NR activity = [(C60 − C0) × V60]/W. where NR activity is expressed in μmol-N g−1 FW h−1, C0, and C60 are the concentration of NO2-N (in μmol ml−1) in the assay medium at t0 and t60, respectively, V60 is the medium volume at t60 (4.5 ml) and W is the Ulva FW (g). 2.6. Biochemical analysis Fig. 2. pH values in the cultivation tanks of Ulva fasciata under the different nutrient regimes: TAN-enriched seawater (TAN-TAN, ●), transfer from TAN- to NO3-N-enriched seawater (TAN-NO3, ), or pre-acclimation in NO3-N-enriched seawater (NO3-NO3, ●). Values are mean ( ± SD), n = 3.

Approximately 200 g of Ulva (FW) were sampled at the beginning and end of the experiment (i.e., days 0 and 3, respectively) for biochemical analysis. C and N content in the biomass were measured using a Flash 2000 CN Elemental Analyzer (Thermo Scientific, Italy). Protein content was estimated by multiplying the total N by a factor of 5.45 [42]. Phosphorus content was determined according to a recommended protocol [43] using 1 M HCL and followed by an ammonium molybdate reaction [44]. Lipid content was measured following a recommended method [45] after a chloroform-methanol extraction during which samples were homogenized for 5 min, separated and vacuum dried.

The mean temperature was 22.1 °C ± 0.2 S.E (ranging between 18 and 27.8 °C). Light intensity during daylight hours ranged between 19 and 1025 μmol photon m−2 s−1 (mean intensity of −2 −1 515 μmol photon m s ± 116 S.E), reaching highest intensity at 1:00 PM (Fig. 1). The pH in the Ulva culture tanks ranged between 8.1 and 9.8 (Fig. 2) and was influenced by the nutrient regime (p < 0.001), with the highest levels in cultures of NO3-NO3 Ulva (9 ± 0.06 S.E), lower ones in TAN-NO3 Ulva (8.9 ± 0.06 S.E; p < 0.01), and lowest when culturing Ulva in TAN (over NO3) as sole nitrogen source (8.7 ± 0.05 S.E; p < 0.001).

2.7. Data analysis Time-series data (i.e., pH, maximum quantum yields, NUR and NR activity) were analyzed using permutation analysis of variances. Significant differences were then determined using pairwise t-tests followed by Bonferroni adjustment of the critical p values. For pair-wise t-tests, maximum quantum yields were arcsin-transformed, NUR and NR activity data were log-transformed, and SD was calculated after back-transformation. Differences in biomass production and composition were detected using one-way ANOVA, where pairwise comparisons were done by post-hoc Tukey's honestly significant difference test. The analysis was performed using R (version 1.1.456 – © 2009–2018 RStudio) with ezANOVA function from “ez” package (version 4.4-0), considering treatment as a between-group factor. For time-series data, time was considered as a within-group factor.

3.2. Biomass production and biochemical composition DGR of Ulva was fastest in cultures of TAN-TAN Ulva (14.3% d−1 ± 1.5 S.E), 28% slower in TAN-NO3 Ulva (10.4% d−1 ± 1.1 S.E), and 48% slower (7.5% d−1 ± 3 S.E, p < 0.05) in the NO3-NO3 Ulva (Fig. 3). N content in Ulva biomass cultured in NO3-N-enriched seawater (TAN-NO3 or NO3-NO3) ranged between 5.3 and 5.8%, significantly lower (p < 0.001) than in the TAN-TAN cultures, where N content ranged between 6.92 and 7.15% (Table 2). While C content was relatively similar in Ulva under different nutrient regimes, a significantly lower C:N ratio of 5.8 ( ± 0.1 S.E) was calculated for Ulva cultures in TAN-enriched seawater compared to 7.1 ( ± 0.1 S.E) in Ulva transferred to or acclimated in NO3-enriched seawater (p < 0.001; Table 2). The NO3-NO3 Ulva contained significantly more lipids (p < 0.05) and phosphate (p < 0.01) compared to the other nutrient regimes. Content of these two components ranged between 1.88 and 2.63% and between 0.33 and 0.46%, respectively (Table 2). Carbohydrate content

3. Results 3.1. Abiotic parameters Temperature, light (Fig. 1) and pH (Fig. 2) revealed daily patterns of low values in the mornings and evenings and peaks during mid-day. 3

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Fig. 4. The maximum quantum yield of Ulva fasciata under different nutrient regimes: TAN-enriched seawater (TAN-TAN, ■), transfer from TAN- to NO3-Nenriched seawater (TAN-NO3, ), or pre-acclimation in NO3-N-enriched seawater (NO3-NO3, □). Values are mean ( ± SD), n = 3.

Fig. 3. Daily growth rate (DGR) of Ulva fasciata under the different nutrient regimes: TAN-enriched seawater (TAN-TAN), transfer from TAN- to NO3-Nenriched seawater (TAN-NO3), or pre-acclimation in NO3-N-enriched seawater (NO3-NO3). Values are mean ( ± SD), n = 3. Letters represent statistical differences at p < 0.05.

3.5. Nitrate reductase activity in the TAN-NO3 Ulva was higher than in the TAN-TAN Ulva (p < 0.05), and similar to that in NO3-NO3 Ulva (p = 0.12) (Table 2). Compared to Ulva cultured in the presence of TAN, ash content was higher in cultures with NO3-N-enriched seawater (p < 0.01; Table 2).

Four hours after reintroduction into NO3-rich seawater, NR activity in cultures of NO3-NO3 Ulva was the fastest (1.95 μmol N g FW−1 h−1 ± 0.2 S.E), followed by a continuous decrease until reaching significantly lower activity (0.57 μmol N g FW−1 h−1 ± 0.1 S.E) 12 h later (p < 0.001) (Fig. 6). During the remainder of the experiment, NR activity in these cultures was between 0.06 and 0.98 μmol N g−1 FW h−1 (Fig. 6). Activation of NR started 28 h after transferring Ulva from TAN- to NO3-N-enriched seawater but, once initiated, the activity of the enzyme increased continuously over the following 36 h until reaching a maximal rate of 2.53 μmol N g−1 FW h−1. Less enzyme activity was measured in the subsequent period but was still faster than in the NO3-NO3 Ulva during the same period (Fig. 6).

3.3. Maximum quantum yields Maximum quantum yield (Fv/Fm) in cultures with NO3 (TAN-NO3 or NO3-NO3 Ulva) was higher than in culture with TAN (TAN-TAN Ulva) (Fig. 4). Pre-acclimation of Ulva in NO3-N-enriched seawater resulted in the highest quantum yield of 0.58 ± 0.02 S.E, compared to Ulva transferred from TAN to NO3-N-enriched seawater (0.54 ± 0.02 S.E; Fig. 4) or to cultures with TAN (0.41 ± 0.07 S.E).

4. Discussion

3.4. Nutrient removal

The current study indicated that the nitrogen source influenced Ulva performance in terms of growth, nutrient uptake and, to some extent, energy metabolism. TAN as the sole N source rather than NO3-N induced increases in biomass production, the specific uptake rate of N, and protein content in the biomass. On the other hand, NO3-N as the sole N source induced faster P uptake and higher photosynthetic activity. NR activity appeared 28 h following the transfer of Ulva from TAN- to NO3-N-enriched seawater, while previous acclimation in NO3N-enriched seawater resulted in lower activity of the enzyme. Overall, photosynthetic quantum yields measured by PAM in the current study for Ulva under the different N regimes are consistent with previous values measured in other green algae, including different Ulva species [46]. The drop in pH from mid-day to the afternoon in Ulva cultured with TAN as sole N source may be attributed to H+ secretion during TAN assimilation [47], but it can also suggest a drop in photosynthetic carbon uptake toward the end of the day [48]. As compared to

Regardless of nitrogen source, the Ulva's nutrient uptake rates on the day of the transfer to new media were 97 μmol N g DW−1 h−1 ± 19 S.E and 11 μmol P g DW−1 h−1 ± 1.2 S.E (Fig. 5). In the following days, nutrient uptake rates dropped (38 μmol g N DW−1 h−1 ± 8 S.E and 7.4 μmol P g DW−1 h−1 ± 0.8 S.E; Fig. 5a, b). TAN-TAN Ulva took up N several-fold faster, 114 μmol N g DW−1 h−1 ± 12 S.E, compared to cultures in NO3-N with a rate of 22 μmol N g DW−1 h−1 ± 5 S.E (p < 0.001; Fig. 5a). In contrast, uptake rate of P by the TAN-TAN Ulva (3.4 μmol P g DW−1 h−1 ± 0.7 S.E) was about a third of the uptake rate in cultures with NO3-N (10.8 μmol P g DW−1 h−1 ± 0.5 S.E) (p < 0.001; Fig. 5b). The uptake rates of N and P by the NO3-NO3 Ulva were similar to their respective values in the TAN-NO3 treatment described above (Fig. 5; p = 1).

Table 2 Biochemical composition of the dried Ulva fasciata cultivated in different nutrient regimes: TAN-enriched seawater (TAN-TAN), transfer from TAN- to NO3-N enriched seawater (TAN-NO3), or pre-acclimation in NO3-N-enriched seawater (NO3-NO3). Content of the various parameters are presented as their percentage of alga dry biomass while C:N ratio was calculated from measurements of the level of these nutrients in the dry biomass. Values are mean ( ± S.E), n = 3. Letters represent statistical differences. ns = not significant.

%C %N C:N % ash %P % lipid % protein % carbohydrate

TAN-TAN

NO3-NO3

TAN-NO3

Level of significance

41.0 ± 0.2 7.1 ± 0.1a 5.8 ± 0.1a 10.1 ± 0.3a 0.17 ± 0.003a 1.65 ± 0.01a 38.6 ± 0.4a 49.7 ± 0.3a

40.6 ± 0.6 5.8 ± 0.03b 7.02 ± 0.1b 14.5 ± 0.8b 0.39 ± 0.04b 2.25 ± 0.2b 31.5 ± 0.1b 51.7 ± 1.1ab

39.2 ± 0.6 5.5 ± 0.1b 7.1 ± 0.1b 14.2 ± 0.5b 0.25 ± 0.01a 1.61 ± 0.1a 30.0 ± 0.8b 54.2 ± 0.5b

ns p < p < p < p < p < p < p <

4

0.001 0.001 0.01 0.01 0.05 0.001 0.05

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Fig. 5. Ulva fasciata specific nitrogen (a) and phosphorus (b) uptake rates under different nutrient regimes: in TAN-enriched seawater (TAN-TAN, ■), transfer from TAN- to NO3-Nenriched seawater (TAN-NO3, ), or pre-acclimation in NO3-N-enriched seawater (NO3NO3, □). Values are mean ( ± SD), n = 3.

rich fishpond effluent [21,64]. Moreover, the faster P uptake of Ulva in NO3-N-rich seawater, as measured in the current study, is consistent with measured P uptake rates of both U. prolifera and U. linza that were cultured with NO3 as sole N source [30,65]. Nevertheless, the measured P uptake by Ulva in NO3-N-rich seawater in the current study surpassed these results despite the higher N:P ratio of 10:1 that was maintained. However, although being taken up rapidly, storage of P by Ulva in NO3-N-enriched seawater is doubtful, as the polyphosphate pools are considered lower in Ulva when fed with NO3-N than with TAN [66]. It is more likely that Ulva utilizes P for the production of phosphorylated, high energy cofactors that are required for the reduction of NO3-N to NH4 prior to assimilation [67,68]. While a previous study suggested a ~24 h period from TAN depletion to initiation of NO3-N uptake by Ulva [8], in the present study NO3-N uptake was immediate. The rapid uptake of N (as either TAN or NO3-N) upon introduction in the culture media is suggested to be the result of the desiccation procedure (i.e., drip drying for 2 h prior to transferring to new media). Such a phenomenon has been described previously, revealing high N content in cells during algal recovery from the desiccation process [69]. Such an accumulation of internal NO3-N typically results in rapid activity of NR afterwards [70], but in the current study NR activity in Ulva that was pre-cultured in TAN was first measured only 24 h after transfer of the Ulva from TAN- to NO3-Nenriched seawater and continued to increase. We assume that despite the immediate luxury uptake of NO3-N, 24 h were required for NR activation in the Ulva but, once initiated, the subsequent increased NR activity was essential to compensate for the high uptake of NO3-N. In the case of previous acclimation in NO3-rich seawater, NR activity was immediate and rapid during the first day but decreased to a relatively constant rate in the subsequent culture period. Following these results, we assume that since NR was already present in cells of Ulva during the pre-acclimation to NO3-N activation was immediate once transferred to similar media with NO3. The decrease in NR activity during first 24 h and its subsequent constant activity are similar to results of previous studies of Ulva cultured in NO3 [68,71]. It can also be suggested that the desiccation of Ulva prior to transfer to new media caused the immediate decrease in NR activity, as also reported in other stressed Ulva, due to high UV radiation [68]. The measured biochemical composition of Ulva under different nutrient regimes may indicate differences in the allocation of carbon in the cells. In a previous study, C allocation to the formation of amino acids was indicated when culturing Ulva with TAN [26]. Since in the current study a faster N uptake was measured in cultures with TAN than in cultures with NO3, it is reasonable to assume that the luxury uptake of N as TAN has led to the greater formation of amino acids and proteins [57]. Since the uptake of NO3 requires greater energy investment for both the active transport into the cells and the subsequent activation of the reducing enzymes NR and NIR, the N taken up in cultures with NO3 is assumed to be at a rate that meets the necessary protein requirement for the algae and hence is lower than that in culture with TAN. Lower N and protein content in Ulva cultured with NO3 as compared to TAN has been reported previously [19]. In cultures with NO3-

Fig. 6. Nitrate reductase activity in Ulva fasciata cultured in NO3-N as the sole N source, after pre-acclimation in either NO3-N-enriched seawater (NO3-NO3, ●) or TAN-enriched seawater (TAN-NO3, ), respectively. Values are mean ( ± SD), n = 3. Asterisks indicate significant level at a certain time point: *p < 0.05, **p < 0.005, ***p < 0.001.

other nutrient regimes, Ulva cultured in TAN-enriched seawater revealed a lower photosynthetic quantum efficiency. Such a phenomenon has been attributed to the toxicity of free NH3 molecules in cultures of microalgae [49,50], as well as in higher plants [51]. The free NH3 molecules can either induce uncoupling of phosphorylation [52,53] or act as a water analog that competes with H2O for binding sites in the Mn complex of PSII [54–56]. Although one can assume a similar effect in the present experiment (i.e., ~0.5 mM of toxic NH3 at the highest measured pH of 9.3), production of Ulva was not decelerated by the apparent lower photosynthetic activity and is consistent with previous production rates of Ulva cultured with different nitrogen sources: TAN and NO3-N [57]. Another possibility is that accumulation of intracellular TAN led to a reductive biochemical state in the chloroplast. Under such conditions photoinhibition with consequently reduced photosynthetic yields may also occur [58]. However, the acute toxicity value of NH3 for marine fish is 0.1 mM (as reported for 17 marine fish species) [59,60], up to five-fold lower than levels in the current study. Therefore, in aquaculture settings, the effluents are unlikely to contain NH3 levels above 0.1 mM and so NH3 is unlikely to negatively impact Ulva photosynthesis [61]. In fact, a previous study revealed that culture of Ulva australis in TAN-enriched media at up to 0.1 mM TAN (compared to ~0.5 mM in the current experiment) improved algae photosynthetic rates, as compared to the ambient level of only 0.4 ± 0.3 μM [62]. Since Ulva in NO3-N-enriched seawater revealed a relatively higher photosynthetic efficiency but slower growth (compared to cultures with TAN), we assume that much of the reductive energy from photosynthesis was utilized for NO3-N reduction by NR and NIR, requiring 137 Kcal mol−1 (i.e., 34 and 103.5 Kcal mol−1 for NR and NIR, respectively) at pH 7 [63]. In contrast to N uptake, P uptake by Ulva when cultured in NO3 as sole N source was up to six-fold faster than in cultures with TAN. The relatively slower uptake of P in cultures with TAN is consistent with previous results from studies of Ulva fasciata or Ulva clathrada in TAN5

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Authors' contribution

N, C content was relatively similar to that in cultures with TAN, but carbohydrate content was higher. The latter may indicate that in cultures with NO3-N the C taken up was primarily allocated for formation of carbohydrates. This is also consistent with the faster photosynthesis in NO3-cultured Ulva, as carbohydrates are major intermediates in the photosynthetic carbon reduction cycle and the photorespiratory carbon oxidation cycle [72]. The faster uptake of orthophosphate in cultures with NO3 in the current study resulted in P- and lipid-rich biomass as compared to cultures with TAN, but according to our results a longer culture with NO3 as sole N source is recommended to achieve maximal P and lipid content as compared to the smaller effect when transferring Ulva from TAN to NO3-N-rich seawater. Considering the potential of Ulva in N removal in fishpond effluent, more attention has been given to the removal of TAN than to NO3-N removal, even though the concentration of NO3-N in mariculture effluents often equals [3,8,73] and even surpasses [74,75] that of TAN. Our findings suggest that Ulva can take up NO3-N, although at a much slower rate than TAN (i.e., 7- to 20-fold slower). The calculated daily uptake rate of NO3-N by the Ulva biofilter in the current study is 34 ± 16 mmol m−2 d−1. This rate corresponds with previous measurements of total oxidized N (i.e., NO2-N + NO3-N) uptake rate of 46 ± 11 mmol N m−2 d−1 by an Ulva biofilter for polishing of mariculture effluents after pre-treatment of fishpond effluent by another upstream Ulva biofilter [19]. While recovery of N from fishpond effluents can be achieved by various bacterial- and plant-based biofilters such as seaweeds, halophytes, and periphyton, P removal by these biofilters is considered negligible [21,73,75,76]. This phenomenon is highly problematic, as documented total P in fishpond effluent has reached 80 mg L−1 [77]. Chemical adsorption has been suggested for P removal from agricultural wastewater [78,79]. However, many of the studied absorbents are considered costly, and recovery of P is questionable [80]. Considering results of the current study, the application of NO3-adapted Ulva for P removal in NO3-rich fishpond effluent is promising.

All authors materially participated in the research and in the manuscript preparation. LG is the corresponding author, research leader and coordinator. BS performed experiments, conducted fieldwork and analyzed data. MS contributed to research design and data analyses. RB, MM and HC contributed to fieldwork. AN, ES and MK contributed to data analyses and writing. References [1] R.H. Piedrahita, Reducing the potential environmental impact of tank aquaculture effluents through intensification and recirculation, Aquaculture 226 (2003) 35–44, https://doi.org/10.1016/S0044-8486(03)00465-4. [2] A.O. Amosu, D.V. Robertson-Andersson, E. Kean, G.W. Maneveldt, L. Cyster, Ulva armoricana (Chlorophyta) in a land-based aquaculture system, Int. J. Agric. Biol. 18 (2016) 298–304, https://doi.org/10.17957/IJAB/15.0086. [3] T. Ben-Ari, A. Neori, D. Ben-Ezra, L. Shauli, V. Odintsov, M. 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CRediT authorship contribution statement Ben Shahar:Investigation, Formal analysis, Data curation, Visualization, Writing - original draft.Muki Shpigel:Methodology, Writing - original draft.Roy Barkan:Formal analysis, Data curation.Matan Masasa:Formal analysis, Data curation, Supervision.Amir Neori:Validation, Writing - original draft, Writing - review & editing.Helena Chernov:Formal analysis.Eitan Salomon:Data curation.Moshe Kiflawi:Data curation.Lior Guttman:Project administration, Supervision, Conceptualization, Methodology, Validation, Writing - original draft, Writing - review & editing.

Acknowledgments The research was supported by the United States - Israel Binational Agricultural Research and Development Fund (BARD) grants No. IS 4995 17R and US 459913 R. The authors thank all the staff at the National Center for Mariculture and in particular A. Zalmanson, D. BenEzra and M. Fedyuk for their invaluable technical assistance. Much appreciation is given to Prof. Maoz Fine's lab team, G. Banc-Prandi, J. Bellworthy and G. Eviatar from the Eilat Interuniversity Institute for Marine Sciences for their support in PAM measurements. The authors also thank Dr. A. Colorni and Mikhal Ben-Shaprut for making useful suggestions to the MS. The authors have no conflicts of interests, financial or otherwise, to declare. No conflicts, informed consent, human or animal rights are applicable.

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