Study on biofiltration capacity and kinetics of nutrient uptake by Gracilaria cervicornis (Turner) J. Agardh (Rhodophyta, Gracilariaceae)

Study on biofiltration capacity and kinetics of nutrient uptake by Gracilaria cervicornis (Turner) J. Agardh (Rhodophyta, Gracilariaceae)

Marcella A. A. Carneiro^I; Fúlvio Aurélio de M. Freire^II; Eliane Marinho-Soriano^I,*

^IDepartamento de Oceanografia e Limnologia, Universidade Federal do Rio Grande do Norte, Brazil

^IIDepartamento de Botânica, Ecologia e Zoologia, Universidade Federal do Rio Grande do Norte, Brazil

ABSTRACT

The absorption efficiency and kinetic parameters (V_max, K_s and V_max:K_s) of the seaweed Gracilaria cervicornis for the nutrients NH₄⁺, NO₃^- and PO₄^3- were evaluated. Absorption efficiency was measured by monitoring nutrient concentrations for 5 h in culture media with initial concentrations of 5, 10, 20 and 30µM. Kinetic parameters were determined by using the Michaelis-Menten formula. Absorption efficiencies for this algae were greater in treatments with lower concentrations, as evidenced by a reduction of 85.3, 97.5 and 81.2% for NH₄⁺, NO₃^- and PO₄^3-, respectively. Kinetic parameters show that G. cervicornis exhibits greater ability to take up high concentrations of NH₄⁺ (V_max=158.5µM g_dw^-1 h^-1) and low concentrations of PO₄^3- (K_s=5µM and V_max:K_s=10.3). These results suggest that this algal species has good absorption capacity for the nutrients tested and may be a promising candidate as a bioremediator of eutrophized environments.

Keywords:Gracilaria, nutrient uptake, macroalgae

Introduction

Seaweeds are particularly efficient nutrient absorbers and possess specific mechanisms for storing large amounts of nitrogen and phosphorous in their tissues (Lobban & Harrison 1997). These nutrients may be used in the future when, for example, the environmental concentrations fall to levels lower than those required by the seaweeds for growth (DeBoer, 1981).

Studies related to nutrient removal by seaweeds have been conducted to select species that can be used as biofilters for the treatment of eutrophized environments (Chopin et al., 2001; Buschmann et al., 2001; Troell et al., 2003; Neori et al., 2004). The seaweeds most widely used in this type of experiment belong to the genus Gracilaria. For example, in an integrated salmon/seaweed system, Buschmann et al. (1996) demonstrated that Gracilaria chilensis was capable of removing 95% of ammonium and 32% of phosphorous. Other integrated studies with Gracilaria gracilis also demonstrated a high capacity for removing these nutrients: around 93% and 62% of ammonium and phosphorous, respectively (Hernández et al., 2002; Martínez-Aragón et al., 2002). More recently, Marinho-Soriano et al. (2009) showed that Gracilaria caudata was capable of reducing the concentrations of ammonium, nitrate and orthophosphate to 59.5, 49.6 and 12.3%, respectively, in only 4 h.

Nutrient uptake rates achieved by seaweeds are generally based on the reduction of the concentration of a given nutrient in the culture medium. In these studies, kinetic parameters (V_max and K_s ) are quite useful for identifying the physiological abilities of a seaweed (Phillips & Hurd, 2004), supplying valuable data to help in the selection of species that can be used as biofilters in eutrophized environments. The aim of the present study was to obtain information on the filtration capacity and the kinetics of uptake of NH₄⁺, NO₃^-and PO₄^3- by the seaweed Gracilaria cervicornis (Turner) J. Agardh.

Materials and Methods

Algal material and preculture conditions

The seaweeds used in this experiment were collected at the Buzios beach in the Northwest of Brazil (06º01'S-35º06'W) and taken to the laboratory, where epiphytes and sediment were removed. Before the experiment, the seaweeds were maintained for 24h in seawater (NH₄⁺<1 µM, NO₃^-<1 µM and PO₄^3-undetectable) with constant aeration and illuminated (180 µmol photon m^-2·s^-1) in a 10 h light-14 h dark cycle at a temperature of 28.6±0.5 ºC and salinity of 37.8±0.2 PSU. The samples used in the absorption experiment were selected from this stock.

Experimental design

The absorption efficiency of G. cervicornis for NH₄⁺, NO₃^-and PO₄^-3 was determined by monitoring these nutrients at initial concentrations of 5, 10, 20 and

30 µM. For each concentration, transparent cylindrical recipients (triplicate) containing 5 g of G. cervicornis and filled with 1 L of filtered seawater were used. To obtain the desired concentrations, previously prepared solutions containing NH₄Cl (NH₄⁺), KNO₃ (NO₃^-) and KH₂PO₄ (PO₄^3-) were added to the seawater. A recipient containing only enriched seawater served as the control. The experiment lasted for 5 h, with samplings at 15 min, 30 min, 1 h, 2 h, 3 h, 4 h and 5 h. To ensure that none of the nutrients (N and P) would become limiting for G. cervicornis, a molar ratio of 10:1 (N:P) was used (Friedlander & Dawes, 1985). Water samples were analyzed according to Strickland & Parsons (1972). After the last sampling, the seaweeds were immediately removed and oven dried (60 ºC) to obtain the dry weight. Absorption efficiency was calculated based on the reduction in the concentration of nutrients for each sampling period and expressed in percentages.

Uptake rates (µM g_dw^-1h^-1) were calculated for each time interval during the depletion according to Pedersen (1994): V=[(S₀ • Vol₀) - (S₁ • Vol₁)]/(B • t), where S₀ and S₁, are the substrate concentrations and Vol₀ and Vol₁, the volumes before and after a sampling period (t), and B is algal dry weight biomass (≈ 0.5 g).

Uptake rates (V) between 0-15 min were plotted against each corresponding mean substrate concentration and the Michaelis-Menten function: V=V_max S/(K_s+S), was fitted to the data by regression, where V_max is the maximum uptake rate (µM g_dw^-1 h^-1) and Ks is the half-saturation constant.

Statistical analyses

All data were tested for normality (Kolmogorov-Smirnov test) and homoscedasticity (Levene's test). One-way ANOVA was used to determine the variations between the time intervals monitored, followed by the Student-Newman-Keuls post-hoc test for multiple comparisons of means (Zar, 1999). The Michaelis-Menten function was analyzed for significance by using the Table Curve^® 2D 5.0, 2000 program. Statistical analyses were conducted with SigmaStat v2.03 software.

Results

Absorption of nutrients by G. cervicornis was significant at all concentrations analyzed (ANOVA, p<0.05), with the lowest values being recorded in the

first 15 min of incubation and the maximum values at the end of the experiment (3-5 h) (Figure 1). The absorption efficiency of G. cervicornis was greatest at the lowest initial concentration (5 µM), showing the greatest percent reduction for NO₃^-(97.5%), followed by NH₄⁺ (85.3%) and PO₄^3-(81.6%). In the treatments with initial concentrations of 20 and 30 µM, higher NH₄⁺ absorption was observed, followed by NO₃ and PO₄^3-. No variations in nutrient concentrations were observed in the controls (ANOVA; p>0.05) (Table 1).

Higher uptake rates (V) were obtained from 0 to 15 min. Uptake rates for the three nutrients decreased over the course of the experiment and are illustrated on a hyperbolic curve (Figure 2). As the external concentration of nutrients increased, the velocity showed a tendency to saturation (Figure 2A, ^B and ^C). Kinetic parameters from the Michaelis-Menten equation obtained for the three nutrients are shown in Table 2. The seaweed G. cervicornis exhibited the highest V_max and K_s values in the experiment with NH₄⁺ (158.5 µM g_dw^-1 h^-1 and 41.6 µM). On the other hand, PO^3- had the lowest V_max and K_s values (51.5 µM g_dw^-1 h^-1 and 5 µM, respectively). The V_max:Ks ratio (10.3) was higher for PO₄^3-.

Discussion

This study showed a significant removal of the three nutrients by the seaweed G. cervicornis, with greater biofiltration efficiency for the two forms of nitrogen than for orthophosphate. The maximum absorption efficiency values obtained in this experiment (85.3% - NH₄⁺; 97.5% -NO₃ and 81.2% - PO₄^-3) were similar and at times higher than data found in the literature. Jones et al. (2001) observed that, in wastewater treatment using seaweeds, Gracilaria edulis was capable of reducing the NH₄⁺ concentration by 74% (2 h), NO₃^- by 97.7% (4 h) and PO₄^3- by 95.1% (10 h). In an experiment using Gracilariopsis longissima, Hernández et al. (2006) recorded mean filtration efficiencies of 93.2% for NH₄⁺ and 62.2 % for PO₄^3- after 7 h of incubation.

With respect to uptake rate, G. cervicornis showed reduced values as the incubation time increased. At the end of the experiment, rates were less than 15% of those obtained at the beginning. This pattern was also observed by other authors for several seaweed species (Pedersen, 1994; Campbell, 1999; Dy & Yap, 2001; Pedersen et al., 2004).

The rate of uptake of NH₄⁺ by G. cervicornis was greater than that of NO₃^- at all concentrations tested. This behavior has been generally observed for a number of seaweed species (D'Elia & DeBoer, 1978; Wallentinus, 1984; Phillips & Hurd, 2004), given that the process of NO₃^- absorption and assimilation is more costly to seaweeds than those for NH₄⁺ (McGlathery et al., 1996).

Maximum uptake rates (V_max) and half-saturation constants (K_s) are generally the most widely used parameters for comparing and contrasting uptake rates in seaweeds. K _s is normally used to estimate the ability of a species to absorb a nutrient at low concentrations, while V_max estimates the maximum uptake rate at high concentrations (Raven & Taylor, 2003). Table 3 shows V and K values for various seaweed species. The V_max va lues for G. cervicornis are higher than those recorded for other species of Gracilaria. The high V_max obtained for NH₄⁺ suggests that G. cervicornis performs better in environments where the concentration is high and where it can uptake NH₄⁺ at a rate proportional to its concentration in the water column. This ability may be an advantage for seaweeds cultivated in eutrophized environments.

In this study, G. cervicornis also exhibited high V_max values for nitrate. This rapid absorption of NO₃ from the medium suggests that this species is highly competitive in removing this nutrient from water. The nitrogen uptake rate was always higher than that of phosphorous at the same concentrations. In relation to PO₄^3-, there are few studies on the uptake kinetics of this nutrient, probably reflecting the fact that orthophosphate concentrations are often near the detection limit (Lobban & Harrison, 1997).

However, seaweeds found in eutrophic environments may exhibit very high K_s and V_max values, such as the species Ulva and Chaethomorpha, which exhibit K_s values of 3.5-10 µM and V_max values of 8.5 - 20.8 µM g_dw^-1 h^-1 (Lavery & McComb, 1991). The high absorption rate recorded in this study for the three nutrients should represent an advantage for this species when nutrient availability in the environment is high. This being so, G. cervicornis can be considered to be an important species for bioremediation in eutrophized environments and in integrated aquaculture.

Acknowledgements

This research was supported by the CNPq and CAPES.

Received 23 Dec 2010

Accepted 22 Jan 2011

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Publication Dates

• Publication in this collection
  06 May 2011
• Date of issue
  Apr 2011

History

• Received
  23 Dec 2010
• Accepted
  22 Jan 2011