Effects of Nitrate Source (Artificial and Fishpond) and UV Radiation on Physiological, Photosynthetic, and Biochemical Parameters of Porphyra dioica for Sustainable Integrated Multitrophic Aquaculture (IMTA)

Abstract

The red macroalga Porphyra plays a key role in Integrated Multi-Trophic Aquaculture (IMTA) systems, acting both as a biofilter and as a source of bioactive compounds (BACs) with nutritional and photoprotective value. This study evaluated how nitrogen source and concentration influence its physiological, photosynthetic, and biochemical responses under ultraviolet radiation (UVR). Gametophytes were cultured for four days under two nitrate sources (artificial and fishpond effluents) at 3 and 5 mM concentrations and exposed to PAR (120 µmol·photons·m⁻²·s⁻¹) and UVR (9 W·m⁻² for 6 h·day⁻¹). Morphological responses, photosynthetic performance, and BACs were quantified. Nitrate uptake increased with nitrate concentration, while growth rate remained unaffected. Samples grown with fishpond effluents, particularly at 3 mM, showed darker pigmentation and higher phycoerythrin and mycosporine-like amino acid (MAA) contents, indicating enhanced nitrogen assimilation and photoprotective capacity. Conversely, 3 mM artificial nitrate in the water promoted the highest electron transport rate and lowest non-photochemical quenching, suggesting greater photosynthetic capacity. Polyphenols and antioxidant activity showed no significant differences among treatments, indicating similar stress status. Overall, it is suggested that fishpond effluents acted as a natural biostimulant, enhancing biliprotein and MAA synthesis without compromising physiological stability, reinforcing its potential for sustainable IMTA-based production of high-value photoprotective compounds.

1. Introduction

Porphyra, a genus of red macroalgae widely distributed in intertidal zones, holds significant ecological, nutritional, and economic importance, particularly in Asian countries, where it is cultivated as a high-value food [1,2,3]. Often referred to as “Nori” when dried and processed, Porphyra species are notable for their rich nutrient profile, including high protein content, essential amino acids, and vitamins [1]. Globally, the annual production of Porphyra sensu lato exceeds 2 million tons (wet weight), representing one of the most valuable seaweed commodities with an estimated market value above USD 2 billion [4]. Although Asia dominates global cultivation, interest in European production has been increasing. In Spain and Portugal, experimental and semi-commercial aquaculture of Porphyra is growing, with national initiatives promoting its valorization for food, cosmetics, and biotechnology applications. At the European level, countries such as Ireland, France, and Norway have also developed pilot farms and processing chains to integrate Porphyra cultivation into sustainable blue economy strategies.

In recent years, increasing attention has been directed toward the bioactive compounds (BACs) present in Porphyra, which exhibit a range of potential health benefits, as antimicrobials, antioxidants, anti-inflammatories, and antivirals [5,6,7,8,9]. Among these BACs, polyphenols and mycosporine-like amino acids (MAAs) stand out due to their antioxidant, anti-inflammatory, and photoprotective properties [10,11,12,13,14,15]. Phenolic compounds in Porphyra contribute to oxidative stress mitigation and may play roles in the prevention of chronic diseases such as diabetes and cancer [13,16]. Meanwhile, MAAs function as natural sunscreens with high antioxidant capacity [17], absorbing ultraviolet radiation (UVR) and preventing UV-induced cellular damage, which underscores their potential in cosmeceutical and pharmaceutical applications [18,19].

Simultaneously, the increasing global demand for sustainable aquaculture practices has led to the development of Integrated Multi-Trophic Aquaculture (IMTA) systems. IMTA involves the co-cultivation of species from different trophic levels, such as fish, mollusks, and seaweeds, in a synergistic setup that enhances nutrient recycling and reduces environmental impact [20]. Worldwide, IMTA is being adopted as a model for circular aquaculture, with active research and pilot sites in Asia, North America, and Europe [21,22]. In Europe, particularly in Spain, industrial IMTA systems have been developed integrating Saccharina latissima with mussel effluents, demonstrating nearly twice the protein content and thereby providing greater added value to the species for both food and feed applications [23]. Although the literature on IMTA and seaweed cultivation remains limited, this topic continues to gain importance in both environmental and economic contexts. As discussed in recent reviews, seaweed-based IMTA presents significant advantages but also faces technical and regulatory challenges. Continued scientific progress, technological innovation, and improved understanding of seaweed value are expected to overcome these barriers [24].

Within IMTA frameworks, Porphyra serves not only as a valuable crop but also as a biofilter, efficiently absorbing excess nutrients, especially nitrogen [25]. Most studies conducted with Porphyra have been carried out under controlled laboratory conditions using nutrient-enriched water, consistently demonstrating its high uptake capacity and resilience to variable nutrient regimes [1,25,26,27,28]. However, direct experimental studies of Porphyra cultivated within fully integrated IMTA systems remain scarce, with existing research primarily highlighting its strong potential and suitability for future IMTA applications. These findings reinforce its role as a promising IMTA candidate species, capable of efficiently recycling nutrients while producing high-value biomass. This dual role of Porphyra, as both a source of high-value BACs and an ecosystem service provider, positions it as a key species in the development of sustainable and economically viable aquaculture systems.

Given its multifaceted benefits, further investigation into the potential of BACs from Porphyra sensu lato cultivated under IMTA could yield valuable insights for optimizing both productivity and functional quality. This study aims to evaluate how the source and concentration of nitrogen, specifically nitrate-derived nitrogen, affect the photosynthetic and biochemical responses of the red macroalga Porphyra dioica.

2. Materials and Methods

2.1. Biological Material

The gametophytes of P. dioica were collected from the rocky shore of Santa Cristina Beach (43°61′ N and 8°18′ W) in Galicia, Spain, in June 2024, by the company Porto Muiños. The algal thalli were transported to the University Institute of Blue Biotechnology and Development (IBYDA) at the University of Málaga (Spain) in plastic bags filled with seawater and placed inside a thermal box. In the laboratory, the thalli were rinsed with diluted artificial seawater (prepared by diluting sea salt from Cádiz, Cádiz, Spain), and healthy portions were selected for acclimation. The selected samples were maintained for 7 days in artificial seawater (32 psu) enriched weekly with 1.5 mM nitrate (NO₃⁻; Fertiberia, Madrid, Spain) and 0.10 mM phosphate (PO₄³⁻; Fertiberia) under controlled conditions: photosynthetically active radiation (PAR, 400–700 nm) of 120 µmol·photons·m⁻²·s⁻¹ (white LED light, 5000 K, 54 W, NU-8416, Nuovo, Singapore), temperature of 15  ±  2 °C, 12 h photoperiod, continuous aeration, and tidal simulation every 12 h (alternating submerged “high tide” and exposed “low tide” phases). After the acclimation period, the biomass was used for the experiment.

Fish specimens of Chelon labrosus were collected from the coastal waters of Málaga Bay under authorization from the Andalusian Government’s Fisheries Agency. They were maintained for more than a year in a 5000 L tank under a natural photoperiod, at 20 ± 4 °C, salinity of 1.0 ± 0.2 psu, and at a density of approximately 5 kg of fish per 1000 L within a recirculating aquaculture system (RAS) equipped with both mechanical and biological filtration. The filtration setup included a 60 L chamber for the physical removal of suspended solids and a UVC lamp for water sterilization. The filtered water then passed into a 200 L biofilter operating under aerobic conditions. The biofilter contained polyethylene biofilm carriers (Acuitec, Spain) in the form of 1 cm cylinders with internal fins that increased the bacterial adhesion surface area to 1180 m²·m⁻³. The biological filtration was based on nitrifying bacteria (Nitrosomonas and Nitrobacter) responsible for converting ammonium (NH₄⁺) to nitrite (NO₂⁻) and subsequently to nitrate (NO₃⁻). The system operated with approximately five complete water renewals per day to ensure efficient circulation and nutrient stabilization. Continuous aeration kept dissolved oxygen at 6.8 ± 0.4 mg·L⁻¹. They were hand-fed twice daily (09:00 and 17:00 h) with a commercial diet (32% protein, 6% fat; TI-3 Tilapia, Skretting, Spain). Importantly, fish were reared during the same seasonal period as algal collection, and the fishpond effluent used in the experiments was collected in June 2024 to match the algal sampling period.

2.2. Algal Cultivation

The culture room conditions were the same as those in the acclimation period. Following acclimation, portions of P. dioica (12  ±  0.05 g) were cultured in 3 L of artificial seawater (32 psu) with artificial nutrients or fishpond effluents for 4 days with UVR (Q-panel lamp, λ = 340 nm) at irradiance of 9 W·m⁻² for 6 h (best combination of exposure time (number of days and UVR hours) observed for MAAs increase in preliminary tests; half of the hours at high tide and the other half of the hours at low tide) (daily dose of 194.4 KJ·m⁻²). In total, four treatments (n = 3), were applied: (1) water with 3 mM of artificial NO₃⁻; (2) fish effluent water (3 mM NO₃⁻); (3) water with 5 mM of artificial NO₃⁻; and (4) fish effluent water (3 mM NO₃⁻) supplemented with an additional 2 mM of artificial NO₃⁻. The 5 mM NO₃⁻ concentration was based on previous studies indicating optimal conditions for MAA production [29], while 3 mM corresponded to the maximum nitrate concentration naturally available in the fishpond effluents. Samples were collected in the fourth day of the experiment two hours after exposure to UVA radiation. In this same moment, photosynthetic parameters were measured, and samples were frozen at −80 °C for BACs extraction.

2.3. Light Microscopy

On the final day of the experiment, fresh samples from all replicates were mounted between a slide and coverslip and examined under a light microscope (Leica DME, Wetzlar, Germany) equipped with a Leica ICC50 W camera. Image processing was performed using LAS-EZ software (version 3.4, Leica). Protoplast length was measured with ImageJ software (version 1.53) using images obtained from light microscopy. Fifty cells were measured per treatment, with one measurement taken per cell.

2.4. Nitrate Quantification: Nitrate Uptake Efficiency (NUE)

Nitrate quantification in the water was performed on the first and last days of the experiment (n = 3). One mL of diluted sample was mixed with 50 μL of Griess reagent (1% w/v sulphanilamide in 1.3 N HCl and 0.005% N-(1-naphthyl)-ethylenediamine dihydrochloride (NED) in Milli-Q water, mixed 1:1) and 100 μL of VCl₃ reagent (2% w/v vanadium (III) chloride in 6 N HCl). The mixture was gently mixed and incubated in a temperature-controlled dry bath at 60 °C for 25 min. After incubation, samples were cooled to room temperature in a water bath, and absorbance was measured at 540 nm [30]. Total nitrate concentration was determined using a nitrate standard curve (1–25 µM, $\mathrm{R}2=0.99;\mathrm{y}=0.0352\mathrm{x}-0.0094$) (Supplementary Figure S1).

2.5. Relative Growth Rate (RGR)

The effect of nitrate source and concentration on the growth of P. dioica was assessed by measuring the difference in fresh biomass between the start and end of the treatments (n = 3). Relative growth rates (RGR) were expressed as the percentage of daily growth and calculated using the formula from [31]:

$$RGR (\% {day}^{-1}) = \left[\right(W_f / W_o)^t^{-1 })] \times 100$$

where W_f is the final fresh weight (g), W₀ is the initial fresh weight (g), and t is the duration of the experiment in days.

2.6. Photosynthesis and Energy Dissipation as In Vivo Chlorophyll A Fluorescence

In vivo chlorophyll a fluorescence was measured in situ, directly under the cultivation conditions inside the culture chambers. Saturating light pulses (>4000 µmol·photons·m⁻²·s⁻¹) were applied to algal thalli (three replicates per cylinder) during the dark phase of the photoperiod. This procedure allowed the quantification of three fluorescence parameters: basal fluorescence (F₀), steady-state fluorescence (F_t), and maximal fluorescence measured in dark-adapted (F_m) or light-acclimated samples (F_m′), using a portable pulse-amplitude-modulated fluorometer (Diving-PAM II Fluorometer, Walz, Germany). The optimal quantum yield (F_v/F_m) and the effective quantum yield (Y_II) were evaluated on the seventh day, and calculated using their respective standard formulas:

$$Fv/Fm={\displaystyle \frac{Fm-Fo}{Fm}}$$

$$Y\left(II\right)={\displaystyle \frac{{Fm}^{\prime }-Ft}{{Fm}^{\prime }}}$$

The electron transport rate (ETR) was determined using rapid light curves (RLCs) by exposing samples to thirteen increasing irradiance levels, ranging from 0 to 1500 µmol·photons·m⁻²·s⁻¹ of actinic red light from the Diving-PAM II. After 30 s of exposure at each irradiance, a saturating pulse was applied. For each irradiance level, several parameters were calculated, including the effective quantum yield (Y_II), ETR, and two types of yield losses: non-photochemical quenching yield $Y\left(NPQ\right)=(F_t/{Fm}^{\prime }) - (F_t/F_m)$, and non-regulated energy dissipation yield $\left(Y\right(NO\left)\right) = F_t/F_m$. Non-photochemical quenching (NPQ) was calculated as $Y\left(NPQ\right)/Y\left(NO\right)$. The energy dissipation rate (EDR) was also evaluated at 124 µmol·photons·m⁻²·s⁻¹ (the irradiance closest to the cultivation condition), 619 µmol·photons·m⁻²·s⁻¹ (midpoint of the RLC), and 1475 µmol·photons·m⁻²·s⁻¹ (maximum irradiance of the RLC). ETR [32] and EDR [33] were calculated as follows:

$$ETR (\mu mol e^{-}·m^{-2}{s}^{-1}) = Y\left(II\right) \times E_{PAR} \times A \times F_{II}$$

$$EDR (\mu mol photons·m^{-2}{s}^{-1})=\left(Y\right(NO)+Y(NPQ\left)\right)\times E_{PAR}\times A\times F_{II}$$

Y(II) represents the effective quantum yield, Y(NO) the yield loss associated with passive thermal dissipation, and Y(NPQ) the yield loss associated with photoregulated processes. E_PAR denotes the incident photosynthetically active radiation (PAR) in µmol·photons·m⁻²·s⁻¹. Absorptance (A) was calculated as $A = 1 - (Et/E_0)$, where E_t is the transmitted irradiance through the thallus and E₀ is the incident irradiance. F_II corresponds to the fraction of chlorophyll a associated with photosystem II, assumed to be 0.15 for red macroalgae [34,35]. The resulting ETR and NPQ versus irradiance curves were fitted according to [36], and the following parameters were derived: photosynthetic efficiency of ETR (αETR), maximum ETR (ETRmax), saturating irradiance of ETR (Ek_ETR), optimal irradiance (Eopt_ETR), NPQ efficiency (α_NPQ), maximum NPQ (NPQ_max), and NPQ saturating irradiance (Ek_NPQ).

2.7. Bioactive Compounds (BACs) Extraction

Bioactive compound (BAC) extractions were performed using 200 mg of fresh algal biomass and 2 mL of extraction solvent (distilled water containing 2.5% sodium carbonate) under alkaline hydrolysis conditions (n = 3). The samples were homogenized for 30 s with an UltraTurrax^® (18,000 rpm) and then extracted at 80 °C for 1.5 h. After extraction, samples were centrifuged at 2721.6× g, and the supernatant was collected. The pH was adjusted to 7.0 using lactic acid (pH meter Horiba LaquaTwin; Kyoto, Japan) [37]. The resulting algal extracts were used for determining the concentrations of bioactive compounds, including phycobiliproteins, polyphenols, and antioxidant activity.

2.8. Total Soluble Phycobiliproteins

Phycobiliprotein concentrations [phycocyanin (PC) and phycoerythrin (PE)] were determined using a UV–visible spectrophotometer (Shimadzu UV-2600) at wavelengths of 498, 615, and 651 nm, following the equations described by [38]. Analyses were performed in triplicate, and results were expressed as milligrams of pigment per gram of dry weight (mg·g⁻¹ DW).

2.9. Total Soluble Polyphenols

Polyphenol analysis was performed using the spectrophotometric Folin–Ciocalteu method as described in [39]. Aliquots of 100 µL of algal extract were mixed with 700 µL of distilled water, 50 µL of Folin–Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA), and 150 µL of 20% sodium carbonate, followed by incubation for 1 h at 4 °C. Absorbance was then measured at 750 nm using a Multiskan FC Microplate Reader (Thermo Scientific™, Waltham, MA, USA). Total phenolic content was quantified using a gallic acid standard curve (1–20 µg·mL⁻¹ considering the dilution in the cuvette; $R^2 = 0.99; y = 0.057x + 0.0311$), where y represents absorbance and x represents phenolic concentration (Supplementary Figure S1). Analyses were conducted in n = 3, and results were expressed as milligrams of gallic acid equivalents per gram of dry weight.

2.10. Antioxidant Activity

Antioxidant capacity was evaluated using the ABTS radical scavenging assay. The ABTS^+• radical cation was generated by mixing 7 mM ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), Sigma-Aldrich) with 2.45 mM potassium persulfate (K₂S₂O₈) in a 0.1 M sodium phosphate buffer (pH 6.5). The mixture was incubated for 16 h in the dark at room temperature to ensure complete radical formation. Before the assay, the ABTS^+• solution was diluted with phosphate buffer until the absorbance at 727 nm reached 0.75 ± 0.05. The reaction was initiated by mixing 950 μL of the diluted ABTS^+• solution with 50 μL of algal extract, following [40]. Samples were gently shaken, and absorbance was measured with a UV–visible spectrophotometer at 727 nm, both at the start (DO_i) and after 8 min (DOf) of incubation. The antioxidant activity (AA%) was calculated using the following formula:

$$AA \% = \left[\right(absDOi - absDOf)/absDOi]\ast 100$$

Antioxidant compound concentrations were determined in n = 3 using a standard curve of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma-Aldrich) ranging from 2 to 10 µg·mL⁻¹ (considering cuvette dilution), with $R^2 = 0.99; y = 15.893x + 2.7335$, where y represents absorbance and x the Trolox concentration (Supplementary Figure S1). Results were expressed as micromoles of Trolox equivalent antioxidant capacity (TEAC) per gram of dry weight (µmol TEAC·g⁻¹ DW).

2.11. Mycosporine-like Amino Acids (MAAs)

For mycosporine-like amino acid (MAA) extraction, 200 mg of fresh algal biomass were mixed with 2 mL of distilled water (n = 3). The mixture was homogenized for 30 s using an UltraTurrax^® (18,000 rpm) and then extracted at 80 °C for 1.5 h. After extraction, samples were centrifuged at 2721.6× g, and the supernatant was collected [40].

MAA quantification followed the method described in [41], with modifications from [42]. One mL of algal extract was filtered through a 0.22 µm membrane prior to chromatographic analysis using an HPLC system (1260 Agilent InfinityLab Series, Santa Clara, CA, USA) equipped with a diode-array detector (DAD). Separation was performed by injecting 10 µL of extract into a C8 Luna column (250 mm × 4.6 mm; Phenomenex, Aschaffenburg, Germany) maintained at 20 °C, with samples kept at 10 °C. The mobile phase consisted of 1.5% aqueous methanol (v/v) and 0.15% acetic acid (v/v) in Milli-Q water, using an isocratic flow rate of 0.5 mL·min⁻¹, with a total run time of 30 min. MAAs were detected at 320 and 330 nm. Isolated MAAs obtained by HPCCC were used as standards [43]. Quantification was based on published molar extinction coefficients (ε) for individual MAAs [44,45], and results were expressed as mg·g⁻¹ dry weight (DW).

2.12. Total Internal Carbon, Nitrogen, and Sulfur

Total internal carbon (C), nitrogen (N), and sulfur (S) contents of freeze-dried and ball-milled algal biomass were determined using a LECO-932 CNHS elemental analyzer (St. Joseph, Kansas City, MI, USA) at the Research Support Central Services (SCAI), University of Malaga, Spain. Analyses were performed in n = 3, and results were expressed as percentages (%).

2.13. Statistical Analysis

Data met the assumptions of normality (Shapiro–Wilk) and homogeneity of variances (Bartlett). Subsequently, a one-way ANOVA was applied, followed by Tukey’s post hoc test (p ≤ 0.05). Statistical analyses were performed in Statistica, Release 7.0. Principal component analysis (PCA) was also conducted, and plots were generated with custom Python scripts executed in Spyder (version 5) to visualize similarities among the physiological variables analyzed.

3. Results

3.1. Effects on Morphology, Cell Length, Nitrate Uptake Efficiency (NUE) and Relative Growth Rate (RGR)

After four days of culture under different nitrate sources and concentrations in the presence of UVR, chloroplasts from samples treated with artificial nitrate (3 or 5 mM) showed slight depigmentation, appearing greenish (Figure 1a,c). In the Photoshop color analysis, the colors were classified as yellowish-green (#908755) and yellowish-neutral (#8F8773), respectively. In contrast, samples exposed to fishpond nitrate retained typical color, brownish thalli (Figure 1b,d). In the Photoshop color analysis, the colors were classified as medium olive-brown (#7A624A) and grayish olive-brown (#71664C), respectively.

At the end of the experiment, protoplast length was highest in samples treated with 3 mM fishpond nitrate, followed by 3 mM artificial nitrate, 5 mM artificial nitrate, and finally 5 mM mixed nitrate (fishpond 3 mM + artificial 2 mM), which showed the shortest cells (

Table 1. Cell length (µm), nitrate uptake efficiency (NUE; %), and relative growth rate (RGR; (% day⁻¹)) in P. dioica after exposure to different nitrate sources and concentrations with UVR for four days (n = 3; mean ± SD). Different letters indicate significant differences according to the one-way analysis of variance, followed by Tukey’s post hoc test (p ≤ 0.05).

Treatment	Cell Length (µm)	NUE (%)	RGR (% day−1)
3 mM Artificial	21.57 ± 4.39 b	5.65 ± 0.95 b	1.84 ± 0.61 a
3 mM Fishpond	23.76 ± 3.35 a	5.57 ± 0.84 b	1.97 ± 0.43 a
5 mM Artificial	18.32 ± 3.19 c	8.25 ± 0.62 a	1.76 ± 0.60 a
5 mM: Fishpond (3 mM) + Artificial (2 mM)	15.98 ± 2.66 d	9.41 ± 0.49 a	1.36 ± 0.33 a

).

Regarding nitrate uptake efficiency, the highest absorption was observed in samples exposed to 5 mM from either source, while both 3 mM treatments exhibited the lowest uptake percentages (Table 1).

Considering the relative growth rate, no statistically significant differences were observed among the treatments.

3.2. Effects on Photosynthesis and Energy Dissipation

Analysis of photosynthetic performance under different nitrate sources and concentrations with UVR exposure revealed significant differences in the ETR at eleven irradiances, as indicated in the statistical table within the graph. At all irradiances where differences occurred, samples treated with artificial nitrate (3 and 5 mM) showed the highest ETR values, whereas natural nitrate (3 mM fishpond and 5 mM mixed: fishpond 3 mM + artificial 2 mM) yielded the lowest. For the ETR curves, nitrate source had the strongest influence (Figure 2a). The NPQ reached its highest value in samples treated with 5 mM artificial nitrate and its lowest in those treated with 3 mM artificial nitrate, with significant differences at six irradiances. For the NPQ curves, nitrate concentration exerted the greater impact (Figure 2b).

For F_v/F_m, α_ETR (photosynthetic electron transport efficiency), and Ek_ETR (saturating irradiance for ETR), no statistical differences were detected among treatments after four days of exposure. By contrast, maximal ETR (ETR_max) was greatest in samples exposed to 3 and 5 mM artificial nitrate. Photoinhibition was observed in all treatments, and the optimal irradiance for ETR (Eopt_ETR) was highest in the 3 mM artificial nitrate treatment, while all other treatments showed less favorable responses (

Table 2. Photosynthetic parameters (optimal quantum yield (F_v/F_m), photosynthetic electron transport rate efficiency (αETR), maximal ETR (ETR_max—µmol·e⁻·m⁻²·s⁻¹), ETR saturated irradiance (Ek_ETR—µmol·photons·m⁻²·s⁻¹), ETR optimal irradiance (Eopt_ETR—µmol·photons·m⁻²·s⁻¹), non-photochemical quenching efficiency (αNPQ), maximal NPQ (NPQ_max), and NPQ saturated irradiance (Ek_NPQ—µmol·photons·m⁻²·s⁻¹), obtained after the adjustment of the rapid light curves (RLCs) in P. dioica after exposure to different nitrate sources and concentrations with UVR for four days. Values are calculated as mean ± standard deviation (SD) (n = 3). Different letters indicate significant differences (p ≤ 0.05).

Treatment	Fv/Fm	αETR	ETRmax	EkETR	EoptETR	αNPQ	NPQmax	EkNPQ
3 mM Artificial	0.60 ± 0.03 a	0.10 ± 0.03 a	4.68 ± 0.21 a	57.80 ± 9.86 a	317.15 ± 46.94 a	0.002 ± 0.0003 c	0.82 ± 0.11 a	339.20 ± 26.55 b
3 mM Fishpond	0.57 ± 0.03 a	0.08 ± 0.01 a	2.79 ± 0.01 b	42.23 ± 10.60 a	126.49 ± 17.36 b	0.004 ± 0.0002 b	0.95 ± 0.23 a	116.76 ± 8.91 c
5 mM Artificial	0.59 ± 0.04 a	0.14 ± 0.02 a	4.46 ± 1.32 a	26.26 ± 5.26 a	227.84 ± 34.75 b	0.003 ± 0.0008 b	0.99 ± 0.15 a	393.08 ± 21.38 a
5 mM: Fishpond (3 mM) + Artificial (2 mM)	0.59 ± 0.04 a	0.09 ± 0.02 a	2.47 ± 0.02 b	27.16 ± 6.24 a	117.77 ± 31.15 b	0.007 ± 0.0003 a	0.96 ± 0.11 a	133.28 ± 18.90 c

). For α_NPQ (non-photochemical quenching efficiency), the largest slope was observed in samples from the 5 mM mixed treatment, whereas the lowest response occurred in samples from 3 mM artificial nitrate. No statistically significant differences were found in NPQ_max among treatments. Regarding the NPQ saturating irradiance (Ek_NPQ), the lowest values were recorded in samples from 3 and 5 mM fishpond nitrate, while the highest was observed at 5 mM artificial nitrate (Table 2).

At 124 µmol·photons·m⁻²·s⁻¹, ETR was highest in the artificial nitrate treatments (3 and 5 mM) and lowest in the 5 mM mixed treatment (fishpond 3 mM + artificial 2 mM), with similarity to the 3 mM fishpond treatment. At this irradiance, EDR was highest in 3 mM fishpond, comparable in the 5 mM mixed treatment, and lowest in 3 mM artificial. The ETR:EDR ratio was highest in the artificial nitrate treatments and lowest in the 5 mM mixed treatment (

Table 3. Electron transport rate (ETR—µmol·e⁻·m⁻²·s⁻¹), energy dissipation rate (EDR—µmol·e⁻·m⁻²·s⁻¹), and ETR:EDR ratio obtained after the adjustment of the rapid light curves (RLCs) at 124, 619, and 1475 µmol·photons·m⁻²·s⁻¹ in P. dioica after exposure to different nitrate sources and concentrations with UVR for four days. Values are calculated as mean ± standard deviation (SD) (n = 3). Different letters indicate significant differences (p ≤ 0.05).

Treatment	ETR124	EDR124	ETR124:EDR124	ETR619	EDR619	ETR631:EDR619	ETR1475	EDR1475	ETR1509:EDR1475
3 mM Artificial	3.77 ± 0.38 a	9.98 ± 0.64 c	0.38	4.01 ± 0.62 a	62.98 ± 1.08 b	0.06	3.00 ± 0.61 a	156.33 ± 41.80 a	0.02
3 mM Fishpond	3.20 ± 0.72 ab	13.30 ± 0.99 a	0.24	1.60 ± 0.48 b	76.72 ± 2.90 a	0.02	0.77 ± 0.24 b	144.24 ± 37.42 a	0.01
5 mM Artificial	4.05 ± 0.92 a	10.88 ± 1.17 bc	0.37	3.64 ± 0.99 a	70.24 ± 4.24 ab	0.05	2.25 ± 0.41 a	116.58 ± 22.42 a	0.02
5 mM: Fishpond (3 mM) + Artificial (2 mM)	2.26 ± 0.36 b	12.45 ± 0.52 ab	0.18	1.47 ± 0.23 b	79.95 ± 6.52 a	0.02	0.84 ± 0.11 b	139.86 ± 9.46 a	0.01

).

At 619 µmol·photons·m⁻²·s⁻¹, ETR remained highest with artificial nitrate and lowest with fishpond nitrate. EDR showed the opposite pattern, being lowest with artificial nitrate and highest with fishpond nitrate. Accordingly, the ETR:EDR ratio was higher with artificial nitrate and lower with fishpond nitrate (Table 3).

At the maximum irradiance, 1475 µmol·photons·m⁻²·s⁻¹, ETR was again highest with artificial nitrate and lowest with fishpond nitrate. EDR did not differ significantly among treatments. The ETR:EDR ratio remained highest with artificial nitrate and lowest with fishpond nitrate (Table 3). As with ETR, nitrate source had the strongest influence on EDR.

3.3. Effects on the Quantification of Photosynthetic Pigments: Phycobiliproteins

After four days of culture under different nitrate sources and concentrations in the presence of UVR, samples treated with 3 mM nitrate from the fishpond showed the highest statistically significant phycoerythrin (PE) content, with values statistically similar to the 5 mM artificial and 5 mM fishpond + artificial nitrate treatment. In contrast, samples treated with 3 mM nitrate from the artificial source exhibited the lowest response. Regarding phycocyanin (PC), the highest statistically significant content was observed in samples treated with 5 mM artificial nitrate, showing statistical similarities with 3 mM fishpond nitrate and 5 mM fishpond + artificial nitrate (Figure 3).

The PE:PC ratio was highest in samples treated with 3 mM nitrate from fishpond and lowest in 5 mM nitrate from artificial source (Figure 3).

3.4. Effects on the Quantification of Polyphenols and on Antioxidant Activity (ABTS Assay)

After four days of culture under different nitrate sources and concentrations in the presence of UVR, no statistical differences in phenolic content or antioxidant activity were observed among the treatments (

Table 4. Concentrations of polyphenols (mg·g⁻¹ dry weight) and antioxidant activity (μmol TEAC·g⁻¹ DW) in P. dioica after exposure to different nitrate sources and concentrations with UVR for four days (n = 3; mean ± SD). Same letters indicate no significant differences (p ≤ 0.05).

Treatment	Polyphenols (mg·g−1 DW)	ABTS (μmol TEAC·g−1 DW)
3 mM Artificial	2.33 ± 0.23 a	158.72 ± 15.66 a
3 mM Fishpond	2.62 ± 0.28 a	157.56 ± 15.75 a
5 mM Artificial	2.70 ± 0.30 a	158.87 ± 11.80 a
5 mM: Fishpond (3 mM) + Artificial (2 mM)	3.02 ± 0.50 a	170.42 ± 14.19 a

).

3.5. Effects on Profiles of Mycosporine-like Amino Acids (MAAs)

In the case of MAAs, two types of MAAs were identified by HPLC: shinorine, and porphyra-334. No statistical differences in shinorine content were detected among treatments. However, Porphyra-334 content was highest in samples treated with 3 mM nitrate from the fishpond, with values statistically similar to 3 mM artificial nitrate. Samples exposed to 5 mM nitrate, either from the artificial source or the mix of artificial and fishpond sources, showed the lowest concentrations of this MAA. Total MAA content (sum of all MAAs detected) was highest in the 3 mM fishpond treatment, followed by 3 mM artificial, then 5 mM (fishpond 3 mM + artificial 2 mM), and lowest in the 5 mM artificial treatment (Figure 4).

3.6. Effects on Total Carbon, Nitrogen and Sulfur

Elemental analysis showed no statistical differences in carbon, nitrogen, or sulfur content among treatments after four days of culture under different nitrate sources and concentrations in the presence of UVR (

Table 5. Concentrations of intern carbon, nitrogen and sulfur (% dry weight) in P. dioica after exposure to different nitrate sources and concentrations with UVR for four days (n = 3; mean ± SD). Same letters indicate no significant differences (p ≤ 0.05).

Treatment	% Carbon	% Nitrogen	% Sulfur
3 mM Artificial	37.74 ± 1.18 a	4.59 ± 0.22 a	1.16 ± 0.16 a
3 mM Fishpond	37.21 ± 2.18 a	4.83 ± 0.59 a	1.16 ± 0.20 a
5 mM Artificial	37.25 ± 2.66 a	4.59 ± 0.44 a	1.02 ± 0.04 a
5 mM: Fishpond (3 mM) + Artificial (2 mM)	37.52 ± 1.06 a	5.19 ± 0.44 a	1.18 ± 0.15 a

).

3.7. Principal Component Analysis (PCA) from P. dioica Photosynthetic and Biochemical Responses Based on Concentration and Source of Nitrate

The PC1 and PC2 explained 55.65% of the variability in the samples across different source and concentrations of nitrate. Considering the nitrate source, a completely displaced group is observed for the samples exposed to 100% fishpond effluent, and a slight displacement for the samples exposed to nitrate from fishpond + artificial, and another group, with an overlapped for the samples exposed to 100% artificial nitrate (Figure 5a). When focusing on nitrate concentration, it can be observed the 5 mM group is tightly clustered, while the 3 mM group is more dispersed and shows some overlap (Figure 5b). Together, the analysis indicates that nitrate source is a major driver of response variation.

4. Discussion

After only four days of exposure to nitrate and UVR, samples treated with 3 mM of nitrate absorbed approximately 5% of the nutrient, while those treated with 5 mM absorbed around 9%. Four days were selected because prior tests with this species by the present group showed maximal MAA production at this duration. Moreover, in red macroalgae exposed to UVR with nitrate enrichment, MAA content declined by day 14 compared with shorter exposure periods [46,47,48,49]. These are relatively low values compared with P. linearis [25], who reported about 95% nitrate uptake at similar concentrations. The main difference between the experiments was in the acclimation phase; in that reference, samples underwent 14 days of nutrient deprivation, whereas in the present study, the algae were acclimated only for 7 days in medium containing 1.5 mM nitrate (the optimal concentration for maintaining Porphyra reported by [25]; thus, no depletion or starvation by nitrate was expected. This prior exposure likely saturated the internal nitrogen reserves, reducing the need for additional uptake. Even so, higher nitrate concentrations (5 mM) still resulted in greater nutrient absorption, consistent with the pattern observed by [50]: the more nitrate available in the medium, the more the algae absorbed.

Growth rate was not affected by either the nitrate source or concentration, unlike what was reported by [51], who observed that Ulva spp., a green seaweed, grown with fishpond-derived nitrate achieved higher growth rates than those supplied with artificial nitrate, and by [52], who reported that Gracilaria vermiculophylla growing near salmon cages showed approximately twice the growth of thalli cultivated at distant sites. On the other hand, [53] observed that green and brown seaweeds (Ulva sp. and Fucus sp.) showed a considerable increase in growth when cultivated near fish farms, whereas the red seaweed (Palmaria sp.) exhibited no significant difference between cultivation in fish farm environments and at distant sites, showing results similar to those of the present study. In the case of Hydropuntia cornea, during the first 7 days, growth rates were comparable with or without fish effluent, but after longer cultivation periods (28 and 42 days), effluent-treated algae displayed a significantly higher growth rate [54]. A longer experimental period might have revealed such differences. Conversely, samples exposed to 3 mM fishpond nitrate exhibited the greatest cell length, while those treated with 5 mM fishpond + artificial nitrate showed the smallest. Typically, a reduction in protoplast length under low nitrate is interpreted as a morphogenetic acclimation response, since smaller cells exhibit a higher surface-to-volume ratio (S/V), improving nutrient uptake and light absorption [55]. However, in this study, the opposite trend was observed: the highest nitrate concentration produced the smallest cells and the greatest nitrate absorption. This suggests that when nitrate availability increases, especially from fishpond sources, cells reduce their S/V ratio.

Thallus color is an economically relevant characteristic, not only for culinary selection of Porphyra strains [56], but also as an indicator of photosynthetic health. Samples supplied with artificial nitrate showed discoloration, whereas those grown with fishpond water, particularly at 3 mM without artificial mix as in 5 mM, displayed a darker and more typical Porphyra sensu lato coloration. This darker color in 3 mM fishpond samples correlates with the higher phycobiliprotein content, particularly phycoerythrin, a nitrogen-rich pigment involved in photosynthesis and known to serve as a nitrogen storage molecule when nitrogen is abundant and as a nitrogen source when it becomes limited [57].

Photosynthetic analyses indicated that samples treated with 3 mM artificial nitrate exhibited the highest electron transport rate (ETR) and the lowest non-photochemical quenching (NPQ), suggesting optimal photosynthetic performance under these conditions. This combination of enhanced ETR and reduced NPQ typically reflects a more efficient use of absorbed light energy for photochemistry rather than for energy dissipation as heat. Interestingly, this treatment did not correspond to the highest growth or concentrations of phycobiliproteins, implying that other pigments or mechanisms could be responsible for the enhanced photosynthetic efficiency observed. Fishpond effluents seem to negatively affect the electron transport rate, reducing maximal photosynthetic capacity; however, this decline has no negative effect on nitrate assimilation, growth rate, or the accumulation of BACs. Thus the decrease in ETR can be an acclimation strategy to the fishpond effluent conditions. In contrast to ETR and Eopt, the other photosynthetic parameters (Fv/Fm, α_ETR, Ek_ETR, NPQ_max) did not differ significantly among treatments, suggesting that the general photosynthetic efficiency was not affected by either the nitrate concentration or source. Nonetheless, samples treated with artificial nitrate showed higher ETR_max values overall, while the 3 mM artificial treatment achieved the highest Eopt_ETR, indicating an optimal balance between light absorption and electron transport at moderate nitrate availability. It is important to note that the relatively high standard deviations observed in some photosynthetic parameters likely reflect natural variability among thalli. Longer experimental periods could reduce this variability by allowing seaweeds to acclimate more fully to the experimental conditions.

Chlorophylls and carotenoids are well-known key metabolites involved in light harvesting and protection of the photosynthetic apparatus [58,59,60]. In particular, carotenoids play a dual role; they capture light energy for photosynthesis and act as antioxidant molecules capable of quenching reactive oxygen species generated under stress [61]. Although these pigments were not quantified in the present study, the best ETR response and the lower NPQ in the 3 mM artificial nitrate treatment strongly suggest that these pigments were active contributors to the observed photosynthetic behavior. The yellowish coloration observed in thalli from artificial nitrate treatments further supports this hypothesis, as increased carotenoid accumulation has been widely associated with photoprotective responses and pigment changes under stress conditions [62,63,64].

Overall, the results suggest that algae supplied with moderate nitrate levels (3 mM) from artificial sources were able to maintain a balanced energy flow between photochemistry and photoprotection. The low NPQ values indicate that these samples experienced minimal excess excitation pressure, implying that the photosystems were operating efficiently and that photodamage was limited. Conversely, higher nitrate concentrations (5 mM) appeared to trigger an increase in NPQ, possibly as a compensatory response to dissipate excess energy or to counteract the formation of reactive oxygen species due to nitrogen-induced metabolic acceleration.

When comparing ETR/EDR ratios across all light intensities tested, artificial nitrate treatments consistently displayed the highest values, revealing a predominance of electron transport over energy dissipation. This indicates that cells from these treatments were capable of efficiently conducting absorbed energy into photosynthetic reactions rather than losing it through thermal processes. Such efficiency could result from well-maintained thylakoid integrity, sufficient energy-sink capacity, or enhanced antioxidant protection from carotenoids and other metabolites [65]. Together, these findings highlight the dynamic interplay between nutrient availability, pigment composition, and energy allocation mechanisms in P. dioica and suggest that moderate nitrate enrichment, especially from artificial sources, can enhance photosynthetic efficiency without inducing severe photoinhibition.

Polyphenols, secondary metabolites typically associated with antioxidant defense and protection against oxidative stress [66], showed no significant variation among treatments, suggesting that all samples were under comparable physiological stress conditions. This finding is consistent with the ABTS antioxidant assay, which also revealed no differences in radical scavenging capacity. This contrasts with findings in Kappaphycus alvarezii grown with fishpond effluents, which showed higher phenolic content and stronger ABTS antioxidant activity [67]. And again, a longer experimental period might have revealed such differences. The lack of variation in internal carbon, nitrate, and sulfur contents further supports the idea that P. dioica maintained a stable biochemical composition across treatments, regardless of nitrate source or concentration. This biochemical balance may indicate that, under the experimental period, P. dioica efficiently regulated its primary metabolism and avoided nutrient-driven metabolic imbalances, even when exposed to differing nutrient regimes and UVR presence. However, longer experimental time might reveal more pronounced metabolic adjustments or delayed responses to nutrient and UVR variations.

In contrast, the synthesis of MAAs, nitrogen-based UV-absorbing compounds with UV-absorbing properties [14,68], responded more dynamically to the treatments. The highest MAA concentrations were observed in samples exposed to 3 mM nitrate, particularly when the nutrient source was fishpond water, even though the statistical difference among treatments was not highly significant. A similar but less pronounced pattern was found at 5 mM, where the presence of fishpond effluent also enhanced MAA accumulation compared to artificial nitrate alone. Consistent with this trend, Hydropuntia cornea cultivated with fish effluent has also been reported to exhibit higher MAA production than cultures grown without effluent [54]. This suggests that other natural nutrient sources may provide co-factors or dissolved organic compounds that stimulate secondary metabolism and photoprotective pathways. Fishpond water, being rich in micronutrients, trace elements, and organic nitrogen compounds derived from aquaculture residues [69,70], may act as a mild biostimulant (micronutrients or organic compounds), promoting the synthesis of MAAs and potentially other nitrogen-containing metabolites, such as phycobiliproteins.

The PCA integrated all physiological, photosynthetic and biochemical variables, summarizing the overall response pattern of P. dioica to different nitrate sources and concentrations. The displacement of the fishpond group along both components indicates that this nutrient source causes changes in nitrogen metabolism and secondary compound synthesis, supporting the hypothesis that aquaculture effluents act as metabolic modulators rather than simple nutrient inputs.

5. Conclusions

This study demonstrates that nitrogen source and concentration distinctly influence the physiological and biochemical responses of Porphyra dioica under RUV. While growth and antioxidant capacity remained stable, secondary metabolism showed clear modulation depending on the nitrate origin. Fishpond effluents, particularly at 3 mM nitrate, enhanced pigmentation and the accumulation of nitrogen-based bioactive compounds such as phycoerythrin and mycosporine-like amino acids (MAAs), indicating efficient nitrogen assimilation and photoprotective capacity. In contrast, artificial nitrate at 3 mM promoted higher photosynthetic electron transport rates and lower non-photochemical quenching, suggesting improved light-use efficiency but reduced pigment accumulation.

These findings suggest that natural nitrate sources from aquaculture effluents can act as mild biostimulants, enriching the biochemical composition of P. dioica without compromising physiological stability. Integrating fishpond-derived nutrients into IMTA systems could therefore reduce fertilizer dependency, close nutrient loops, and enhance the production of high-value metabolites with potential applications in nutraceutical and cosmeceutical sectors.

Future IMTA development should explore long-term cultivation trials combining Porphyra with different fish species and effluent compositions to optimize nutrient recycling and the yield of targeted bioactive compounds.