Introduction

There is growing concern on the environmental impacts and eutrophication caused by marine aquaculture and by other human activities as discharge of effluents into seawater. Seaweeds are efficient in the uptake of nitrate, ammonium, and phosphate from seawater, and the assimilation of these nutrients into nitrogenous compounds (e.g., amino acids, proteins, pigments) stimulates seaweed growth (Lobban and Harrison 1997). Therefore, seaweeds could be used as biofilters to remediate the eutrophication, and their cultivation has an essential role in the integrated multi-trophic aquaculture systems (Neori 2008; Buschmann et al. 2008; Bolton et al. 2009).

There are some reports on the role of seaweeds as biofilters for nutrient removal in integrated cultivation, mainly with species of the genera Ulva and Gracilaria; for example, integrated cultivation of salmon and Gracilaria chilensis (Buschmann et al. 2008); fish and Ulva rotundata, Ulva intestinalis, and Gracilaria gracilis (Martinez-Aragón et al. 2002); Ulva lactuca and abalone Haliotis midae (Robertson-Anderson et al. 2008); and Gracilaria lemaneiformis and scallop Chlamys farreri (Mao et al. 2009). There are few reports on integrated cultivation of brown algae: Laminaria saccharina and salmon (Subandar et al. 1993) and Macrocystis pyrifera and salmon (Buschmann et al. 2008).

In Brazil, there are some studies on nutrient removal by seaweeds in integrated cultivation, as for example cultivation of shrimp and Ulva lactuca (Costa 2006), shrimp and Gracilaria cervicornis (Carneiro 2007), fish (Trachinotus carolinus) and Kappaphycus alvarezii (Hayashi et al. 2008), and shrimp and Gracilaria birdiae (Rocha 2009). However, G. birdiae had the capability to remove only nitrite and ammonium from seawater, but did not remove phosphate, which was lethal to this species (Rocha 2009).

The red alga Hypnea musciformis has potential as a biofilter with efficient nitrogen and phosphorus uptake and further assimilation into pigments and proteins (Haines and Wheeler 1978; Martins et al. 2009, 2011). However, ammonium concentrations higher than 15 μM were toxic and lethal for four strains of H. musciformis (Martins and Yokoya 2010). Therefore, the selection of seaweed species for their use as biofilters should be based on the knowledge of their nutrient requirements and tolerance to wide variations of nutrient concentrations. Besides the physiological capabilities for growth and removal of dissolved nutrients, the commercial potential of the seaweed species is also important (Buschmann et al. 2008; Abreu et al. 2011). From this point of view, Hypnea cervicornis (Rhodophyta, Gigartinales) could be a good candidate because it has economic importance being raw material for carrageenan production. During the last two decades, some studies have reported the biological activities of carrageenan, as antiviral activities against HSV and HIV viruses (Neushul 1990; Knutsen et al. 1995; Rodrigues et al. 2011). Moreover, Hypnea spp. produce lectins, glycoproteins used as an anti-inflammatory in cancer treatment (Nagano et al. 2005), as antibiotics (Nascimento et al. 2006), and antifungal activities against human pathogenic yeasts (Cordeiro et al. 2006).

The aim of this study was to evaluate the tolerance and the physiological capabilities of H. cervicornis to growth under a wide range of nitrate, ammonium, and phosphate availabilities and to assimilate them into soluble proteins and photosynthetic pigments.

Material and methods

Species studied and unialgal cultures

Tetrasporophytes of Hypnea cervicornis J. Agardh (Rhodophyta, Gigartinales) were collected at Fortaleza beach, Ubatuba, southeastern of Brazil. Voucher specimens were deposited in the Herbarium of Instituto de Botânica (SP), with the accession number SP 390904. Unialgal cultures were obtained by vegetative propagation of thallus segments of 1 cm in length. Culture medium was composed of sterilized seawater enriched with 25 % of von Stosch solution (VSES/4) following Edwards (1970), with vitamin concentrations reduced to 50 % following Yokoya (2000). Medium renewal was carried out every week. Culture conditions were comparable to those observed in the field: temperature of 23 ± 3 °C, salinity 30 psu, pH 8.0, under irradiances of 60–90 μmol photons m−2 s−1 provided by cool-white fluorescent lamps with a 14:10-h light/dark cycle.

Experiments with ammonium, nitrate, and phosphate

Treatments were composed of sterilized seawater enriched with 25 % of von Stosch solution prepared without nitrogen and phosphorus (VSES/4 modified), but with salts of iron, manganese, EDTA, and three vitamins (Yokoya 2000). The absence of these compounds could inhibit algal growth, so this culture medium was adopted. Inorganic nitrate (NaNO3) or ammonium (NH4Cl) and phosphate (Na2HPO4·12H2O) were added to the VSES/4 medium modified in combinations to obtain ratios of nitrogen/phosphorus (N/P) of 100:1 and 10:1. Sterilized seawater without addition of any nutrients was also tested (named H2O). Concentrations of nitrate and ammonium added to the medium varied, respectively, from 0 to 500 μM and from 0 to 50 μM. Phosphate concentrations varied from 0 to 50 μM when combined with nitrate and from 0 to 5 μM with ammonium. Experiments were conducted at the same conditions of unialgal cultures. The following variables were analyzed: growth rate, protein, and pigment contents. Each treatment was tested with six replicates of five apical segments cultured in glass flasks with 80 mL culture medium. At the end of the experimental period (28 days), culture medium was renewed, and after 4 days (32nd day), samples were frozen with liquid nitrogen and stored at −80 °C for biochemical analysis. This procedure was adopted to standardize the period between culture medium renewal and sample freezing because this factor affects the protein and pigment contents.

Growth rates

Fresh biomass was recorded weekly for 4 weeks, at the same intervals of medium renewal. Growth rates (GR) were calculated from six replicates of each treatment and calculated as [ln (Bf ⋅ Bo−1) ⋅ t −1], where Bo is the initial fresh biomass, Bf is the fresh biomass after t days, and t corresponds to the experimental period (Yokoya et al. 2003).

Pigment contents

The algal mass (80 mg fresh mass for each replicate, n = 3) was ground to a powder with liquid nitrogen and mixed with 50 mM phosphate buffer (pH 5.5). The homogenates were centrifuged at 14,000×g for 20 min., at 4 °C, in order to separate the phycobiliproteins present in the supernatant. Chlorophyll a was extracted after dissolving the pellet in 90 % acetone and centrifuging at 12,000×g for 15 min., at 4 °C. Pigments were quantified by spectrophotometry, and concentrations were calculated following Kursar et al. (1983) for phycobiliproteins (phycoerythrin—PE, phycocyanin—PC, and allophycocyanin—APC) and following Jeffrey and Humphrey (1975) for chlorophyll a (Chl a).

Total soluble protein contents

The algal biomass (80 mg fresh mass for each replicate, n = 3) was ground with liquid nitrogen, and extractions were carried out at 4 °C using 0.2 M phosphate buffer (pH 8) containing 5 mM EDTA and 1 mM DTT. Buffer was added in the proportion of 10 mL g−1 fresh biomass and the homogenates were centrifuged at 12,000×g for 15 min. Total soluble protein contents were determined according to Bradford (1976), using a Bio-Rad protein assay kit and BSA as standard.

Statistical analyses

Data were analyzed by one-way analysis of variance (ANOVA), followed by Student–Newman–Keuls multiple comparison tests and were conducted to distinguish significantly different results (p < 0.05). Statistical tests were performed using STATISTICA software (version 9).

Results

H. cervicornis tolerated all the treatments up to 50:5 μM of ammonium/phosphate and 500:50 μM of nitrate/phosphate (Fig. 1a, b). GR increased proportionally with ammonium addition, and the highest GR was observed in treatments with 50 μM of ammonium with the N/P ratio of 100:1 (Fig. 1a). The lowest GR was observed in treatment with seawater without nutrient addition (H2O), and in concentrations higher than 20 μM of ammonium, GR were higher in treatments with the N/P ratio of 100:1 than 10:1 (Fig. 1a). With nitrate addition, growth rates showed saturation at the addition of 100 μM nitrate for the N/P ratio of 10:1 and 200 μM nitrate for the N/P ratio of 100:1 (Fig. 1b).

Fig. 1
figure 1

Growth rates of H. cervicornis cultured for 28 days in sterile seawater enriched with different ammonium (a) and nitrate (b) concentrations. Each data point is the mean of six replicates and bars are standard deviation. Treatments with distinct letter indicate significant differences according to the one-way ANOVA and Student–Newman–Keuls multiple comparison test (p < 0.05). Empty bars N/P 10:1 and filled bars N/P 100:1

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The content of total soluble proteins in H. cervicornis was higher (approximately threefold) in algae cultured with nitrate addition than with ammonium addition (Fig. 2a, b), and protein concentrations were higher in treatments with high phosphate concentration with ammonium (10:1 N/P) and with low phosphate concentration with nitrate (100:1 N/P). In algae cultured with ammonium, the highest protein concentration was observed in treatments with addition of 50 μM in the N/P ratio of 10:1 (Fig. 2a). Protein contents increased with the addition of nitrate in the N/P ratio of 10:1 (Fig. 2b); however, protein concentrations did not vary significantly in treatments with addition of 100 to 500 μM nitrate in the N/P ratio of 100:1.

Fig. 2
figure 2

Concentration (mg g−1 fresh weight) of soluble proteins of H. cervicornis cultured for 32 days in sterile seawater enriched with different ammonium (a) and nitrate (b) concentrations. Each data point is the mean of three replicates and bars are standard deviation. Treatments with distinct letter indicate significant differences according to the one-way ANOVA and Student–Newman–Keuls multiple comparison test (p < 0.05). Empty bars N/P 10:1 and filled bars N/P 100:1

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Ammonium assimilation in H. cervicornis was mainly in the form of allophycocyanin and phycoerythrin (Fig. 3a). The contents of APC, PC, and PE were higher in the treatments with additions of 30 μM ammonium in N/P ratios of 10:1 and 100:1 (Fig. 3a). The lowest content of APC was observed in seawater, control, and addition of 10 μM ammonium in the N/P ratio of 10:1. The lowest contents of PC and PE were in seawater, control, 10 μM in 10:1 and 100:1 N/P ratio, and with addition of 20 μM ammonium in the N/P ratio of 10:1 (Fig. 3a). On the other hand, nitrate assimilation was mainly in the form of PE (Fig. 3b). Phycobiliprotein concentrations were lower in the control and seawater, and contents of APC and PC were higher in treatments with addition of 400 μM nitrate in the N/P ratio of 10:1 (Fig. 3b). PE contents were below the limit of detection in treatments without nutrient additions (seawater and control), and the highest content was observed in treatments with 100 to 500 μM nitrate in the N/P ratio of 100:1 and 400 and 500 μM nitrate in the N/P ratio of 10:1 (Fig. 3b).

Fig. 3
figure 3

Concentrations (mg g−1 fresh weight) of allophycocyanin, phycocyanin, and phycoerythrin of H. cervicornis cultured for 32 days in sterile seawater enriched with different ammonium (a) and nitrate (b) concentrations. Each data point is the mean of three replicates and bars are standard deviation. Treatments with distinct letter indicate significant differences according to the one-way ANOVA and Student–Newman–Keuls multiple comparison test (p < 0.05). Empty bars N/P 10:1 and filled bars N/P 100:1

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Contents of Chl a in H. cervicornis were lower in treatments with seawater, control, and 10 and 20 μM ammonium in both N/P ratios (Fig. 4a). The highest content of Chl a was observed in treatments with additions of 30 to 50 μM ammonium in the N/P ratio 10:1 (Fig. 4a). The highest content of Chl a was observed in treatments with addition of nitrate and phosphate in the N/P ratio of 10:1, but differences were not significant among these treatments (Fig. 4b).

Fig. 4
figure 4

Concentration (mg g−1 fresh weight) of chlorophyll a (Cl a) of H. cervicornis cultured for 32 days in sterile seawater enriched with different a ammonium concentrations and b nitrate concentrations. Each data point is the mean of three replicates and bars are standard deviation. Treatments with distinct letter indicate significant differences according to the one-way ANOVA and Student–Newman–Keuls multiple comparison test (p < 0.05). Empty bars N/P 10:1 and filled bars N/P 100:1

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Discussion

Growth rates of H. cervicornis increased proportionally with the enhancement of ammonium availability. H. musciformis showed higher uptake in concentrations of 60 μM ammonium in the medium (Haines and Wheeler 1978). On the other hand, ammonium concentrations higher than 15 μM inhibited the growth of brown and light green strains of H. musciformis (Martins and Yokoya 2010). The ammonium toxicity may be a consequence of the lack of carbon skeleton needed for its assimilation, and ammonium accumulation in the cytoplasm generates protons, decreasing the cytoplasmatic pH. Similar results were also observed for the red algae Neoagardhiella baileyi (Kützing) Wynne & Taylor and Gracilaria foliifera (Forskåll) Børgesen, which showed saturation kinetics with addition of 20 μM ammonium (DeBoer et al. 1978).

Growth rates of H. cervicornis showed saturation kinetics in nitrate and phosphate, reaching maximum values with additions of nitrate concentrations higher than 100 μM, and phosphate availability stimulated higher growth rates and saturation occurred in lower concentrations. This is also reported in the color strains of H. musciformis, and growth reaches the saturation at 125 μM of nitrate (Martins and Yokoya 2010). Porphyra torta Krishnamurthy showed higher growth rates with addition of 88 μM nitrate (Conitz et al. 2001), while Gracilaria pacifica Abbott had higher growth in addition of 1 mM (Naldi and Wheeler 1999).

Total soluble proteins can be the largest pool of nitrogen storage in seaweeds and phosphate availability increases the assimilation of nitrogen such as proteins, as reported in Gelidium crinale (Turner) Gailloon in addition of 50 μM ammonium and 10 μM phosphate (N/P ratio of 5:1) (Perfecto et al. 2004). G. pacifica (Naldi and Wheeler 1999) and Gracilaria tikvahiae McLachlan (Bird et al. 1982) showed higher protein content in additions of 1 mM ammonium, while G. chilensis Bird, McLachlan, & Oliveira had higher protein content in low concentrations of ammonium (0.02 μM) and phosphate (0.51 μM) (Buschmann et al. 2008). Gracilaria sp. showed higher protein contents with addition of 75 μM nitrate in the N/P ratio of 37:1 (Andria et al. 1999). These data corroborate with the results described in the present study.

As observed in ammonium assimilation in H. cervicornis, the main form of nitrate assimilation occurred as total soluble proteins and phycobiliproteins, mainly at the form of phycoerythrin. The higher phosphate availability did not influence this response in H. cervicornis. Similar results were also reported in H. musciformis, which showed high protein contents when nitrate availability increased, but phosphate addition did no alter this response (Martins et al. 2009, 2011).

H. cervicornis incorporated ammonium as phycobiliproteins, and allophycocyanin and phycoeryhrin were the main nitrogen storage. Under nitrogen and phosphate limitation, phycoerythrin concentrations were low because this pigment was preferentially metabolized to provide nutrients to sustain the algal growth. Contents of chlorophyll a were low and its accumulation was stimulated by high phosphate availability. Studies of the red alga G. tikvahiae indicated that chlorophyll a did not contribute greatly to the overall nitrogen accumulation (Bird et al. 1982), and this species showed higher nitrogen assimilation as phycoerythrin. This is also observed in Porphyra linearis Greville, Porphyra umbilicalis Kützing, Porphyra leucosticta Thuret, and Porphyra amplissima (Kjellman) Setchell & Huss, which assimilate nitrogen as phycoerythrin, and this process was stimulated with addition of 250 μM ammonium in the N/P ratio of 10:1 (Kim et al. 2007). Nitrate was assimilated as phycocyanin and phycoerythrin by Porphyra dioica Brodie & Irvine with the increase of nitrate availability, and phycoerythrin was the main form of nitrogen pool (Pereira et al. 2008).

H. cervicornis tolerated the highest ammonium and nitrate concentrations (50 and 500 μM, respectively), and accumulated the excess of nitrogen as proteins and phycobiliproteins under higher phosphate availability (N/P ratio of 10:1). These physiological capabilities suggest that this species could be used as biofilter for nutrient removal in eutrophicated seawater and could be cultivated in integrated multitrophic aquaculture systems.