Phosphatase activity in salt-ponds of the Bay of Cádiz
Actividad fosfatásica en salinas de la Bahía de Cádiz
M.D. Frutos1, J. Blasco2* and A. Gómez-Parra1
1 Departamento de Química-Física. Facultad de Ciencias del Mar, Universidad de Cádiz Apartado 40 11510 Puerto Real (Cádiz), Spain.
]]> 2 Departamento de Oceanografía Instituto de Ciencias Marinas de Andalucía (CSIC). Campus Univ. Río San Pedro 11510 Puerto Real (Cádiz), Spain. * E-mail: julian.blasco@icman.csic.es
Recibido en abril de 2003;
aceptado en marzo de 2004.
Abstract
Acid and alkaline phosphatase activities (E.C.3.1.3.2 and E.C.3.1.3.1, respectively) were measured in seawater and sediments from different sites of a salt-pond of the Bay of Cádiz. The profiles of enzymatic activity relative to depth were also analysed. Initially, the optimum conditions (pH, temperature, substrate concentration and amount of sample) for determining enzymatic activities were studied. The apparent activation energies for alkaline phosphatase activity in seawater and sediments were 53.8 and 48.6 kJ mol-1, respectively. The Km and Vmax were 10.6 mM and 89 µg of 4-nitrophenol g-1 h-1 and 8.42 mM and 145 µg of 4-nitrophenol L-1 h-1 for sediments and seawater, respectively. Both types of enzymatic activity reached high values in seawater and sediments; in the confined areas of the salt-pond the values were around 100 µg of 4-nitrophenol L-1 h-1 for seawater and higher for sediments. In general, clear seasonal evolutions for both types of enzymatic activity were found in the seawater, with maxima at the end of summer and minima in winter. No seasonal pattern was evident in the sediment. Stratification of phosphorus regeneration was observed in the sediment column but there was no stratification at depths greater than 15 cm.
Key words: phosphatases, seawater, sediment, salt-ponds, Bay of Cádiz.
Resumen
]]> Se midieron las actividades fosfatásicas ácida y alcalina ((E.C.3.1.3.2 y E.C.3.1.3.1, respectivamente) en muestras de agua y sedimento en diferentes estaciones de salinas de la Bahía de Cádiz. También se analizaron los perfiles de actividad fosfatásica con relación a la profundidad. Se estudiaron las condiciones óptimas (pH, temperatura, concentración de sustrato y cantidad de muestra) para la determinación de las actividades enzimáticas. La energía de activación aparente para la actividad fosfatásica alcalina en agua y sedimento fueron 53.8 y 48.6 kJ mol-1, respectivamente. Los valores de Km y Vmax fueron 10.6 mM y 89 µg de 4-nitrofenol g-1 h-1 y 8.42 mM y 145 µg de 4-nitrofenol L-1 h-1, para sedimentos y agua, respectivamente. Ambos tipos de actividades enzimáticas presentan elevados valores en agua y sedimentos; en las áreas más confinadas de las salinas, los valores alcanzados llegan a ser del orden de 100 µg de 4-nitrofenol L-1 h-1 en las muestras de agua de mar, y superiores en los sedimentos. En general, se apreciaron evoluciones estacionales para ambos tipos de actividades fosfatásicas en las muestras de agua, con máximos a finales del verano y mínimos en invierno. En los sedimentos no se observó esta estacionalidad. La estratificación en la regeneración del fósforo es apreciable en la columna del sedimento, si bien ésta no se observa a profundidades mayores de 15 cm.Palabras claves: fosfatasas, agua de mar, sedimentos, salinas, Bahía de Cádiz.
Introduction
Littoral ecosystems receive large inputs of organic and inorganic materials of lithogenic and anthropogenic origin that tend to accumulate in these areas. Organic matter is the substrate of heterotrophic microbial activities, which results in the release of phosphorus and nitrogen into the environment (Sandstrom, 1982). Nutrient regeneration is the main mechanism of fertilisation in some ecosystems such as the salt marshes around the Bay of Cádiz. These systems are characterised by their high productivity and limited exchange of seawater (Establier et al., 1984).
Potential phosphatase activities are a measurement of the nutritional status of ecosystems (Berman, 1970; Jones, 1972; Perry, 1972; Huber and Kidby, 1984a). The main advantage over other measurement techniques is the large number of samples that can be processed with moderate analytical support. This approach is of special interest in the study of ecosystems with microspatial variations in the physicochemical characteristics of the water column and sediment, for which many sampling stations must be surveyed.
The aim of this study is to determine the potential regeneration rate of inorganic phosphorus and its seasonal and spatial variation in the Bay of Cádiz, differentiating the contribution of each environmental compartment (seawater and surface sediments). Aspects related to the methodology and the optimum conditions (pH, temperature, substrate concentration and amount of sample) for determining the acid and alkaline phosphatases in seawater and sediment were also studied.
Materials and methods
The salt-pond selected for this study is located in the southern part of the Sancti-Petri Channel, at a distance of 4 km from the Atlantic Ocean. It has a surface of 30 ha (fig. 1) and three floodgates to control the seawater exchange, allowing water to enter the salt-pond when the height of the tide exceeds 1.5 m. As a consequence of its location, the pond is not greatly affected by urban sewage, and the quality of the water as well as fish productivity are high (Blasco et al., 1987).
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For this study of the spatial and seasonal variation of both types of phosphatase activity in seawater and sediment, five sampling stations were selected. Station 1 was situated in the outer part of the salt-pond, and stations 2 to 5 were in the inner area (fig. 2). Station 2 was located in the deeper zone (at 1.5 m depth), while stations 3, 4 and 5 were at sites with a depth of about 0.5 m. At all stations, water samples were collected every 10 days and sediment samples monthly, over a period of 12 months. The samples of seawater were collected with sterilised glass bottles. Immediately after collection, the enzymatic activities were analysed according to the procedure described by Hayashi (1972), with several modifications: volumes of 25 to 150 mL of water were filtered through a sterile filter of 0.45 µm pore size (Millipore HAVPO4700); the filters were then used as inoculate for the culture medium (5 mL of 15 mM pNPP and 18 mL of buffer prepared with seawater). To determine the optimum pH conditions, buffer 0.1 M citric acid-sodium citrate, pH 3.0-7.0, and 0.1 M Tris-HCl, pH 7.5-10.0, were employed. The effect of substrate concentration on enzymatic activities was analysed in the range of 1-50 mM. The incubation time ranged from 12 hours to 8 days, when the effect of incubation time was assayed, and the temperature from 5°C to 70°C. The reaction was stopped by adding 5 mL of saturated sodium carbonate and 3 mL of 3% EDTA. The absorbance was read at 405 nm (Reichardt et al. , 1967) and the results were expressed as µg of 4-nitrophenol released L-1 h-1. Salinity was measured using a salinometer (Grundy 6230N). Phosphate determination was performed in a Technicon Traacs 800 Autoanalyser. The extraction of pigments and their quantification was carried out with 90% aqueous acetone, according to the procedure described by Parson et al. (1984).
The sediment samples were collected with a PVC core of 40 mm inner diameter and were maintained at -18°C until processing. Assays previously carried out showed that freezing did not affect the phosphatase activity of the sediment. When the samples were thawed, slices corresponding to the different depths of the sediment were selected. Sections from the first 3 cm, corresponding to surface sediment at all sites, and sections corresponding to 6-9, 12-15, 18-21 and 24-27 cm depth were also taken. These were homogenised by mechanical agitation for 30 min with 100 mL of sterile saline solution (36 g L-1).
The amount of inoculate employed to determine phosphatase activity was 1 mL of suspension. Aliquots of 5 mL of suspension and saline solution, in duplicate, were dried at 80°C until constant weight; these were used to determine the amount of dried sediment needed as inoculate. The results are expressed as µg of 4-nitrophenol released h-1 g-1. The general conditions (pH, time, temperature and substrate concentration) to determine acid and alkaline phosphatase are reported in table 1.
The Kruskal-Wallis comparison of means test (Ruiz-Maya, 1977) was used for the analysis of the differences between sampling stations and seasons.
]]> Results and discussion
Analytical conditions
From a review of the bibliography, the analytical conditions for measuring potential enzymatic activities showed a wide variability depending on the location and sampling season (table 2). This can be due to differences between the species responsible for these activities (phytoplankton and bacterioplankton) and the season when the study was carried out.
The influence on the phosphatase activities of several variables in water and sediment was studied. The variables selected were: pH of culture medium, inoculate quantity, substrate concentration, temperature and incubation time.
pH of incubation medium
Figure 3 shows the variation of acid (AcPA) and alkaline (APA) phosphatase activity in terms of pH for samples from different sites in the Bay of Cádiz in different seasons.
Over the ranges shown, the pH was tested every 0.5 units. The effect of pH on phosphatase activity showed considerable variation between the samples, especially for sediment. This is a consequence of the influence of environmental conditions and the diversity of microorganisms and, therefore, of the particular enzymes responsible for phosphatase activities. Similar results have been reported by other authors (Berman, 1970; Huber et al., 1984a). Hayashi (1972) found that, for marine bacteria, maximum phosphatase activity occurred between pH 5.6 and 9-10. Kuenzler and Perras (1965) obtained maximum activity in the range pH 4.4-9.8 for 16 algal species.
Nevertheless, the majority of assays showed the highest values of APA at pH 8.5, while for AcPA this value was between pH 5 and 6. These optimum pH levels obtained are similar to those reported by other authors (table 2) as suitable for estimating potential phosphatase activity.
Influence of inoculate quantity and incubation time
]]> The influence of inoculate quantity and incubation time is shown in figures 4 and 5. For both variables, linear responses were found for potential phosphatase activity over a wide range of volumes of filtered seawater, sediment and assay times.With regard to the influence of incubation time on phosphatase activity, a decrease in the rate of substrate degradation was observed from the sixth day of the assay. This is related to the increased concentration of the products released during the reaction (phosphate exerts an inhibition effect on the use of organic phosphorus; Siuda, 1984); 4-nitrophenol, which is very toxic for organisms that synthesize phosphatases, is also increasingly present.
On the other hand, the acclimation period of the organisms to the culture medium is very short. In the case of seawater this is less than one day and for the sediment it is even less, as can be observed in figure 5.
Influence of incubation temperature
The effect of incubation temperature was studied in the range of 30-70°C. The results for water and sediment are plotted in figure 6(a, c). An increase in maximum phosphatase activity in seawater was found in the 30-40°C range, with enzymatic activity decreasing as the temperature increases beyond this as a consequence of thermal denaturalisation of the enzyme molecules. This effect can be clearly observed when the temperature coefficient Q10 is plotted as bars in the figures.
The mean value of Q10 for the sediments (fig. 6c) in the range of 30-60°C is 2.01, while for the water (fig. 6a) it is less (1.57). In seawater, the analysed range is 30-50°C, so when the temperature increases above this level, the activity decreases. These findings suggest that the enzymes involved in phosphatase activity in the sediment are more sensitive to temperature than the seawater enzymes. The values reported for these parameters in sediments were similar to the values found by Frankenberger and Tabatabai (1980) for other soil enzymes (aminohydrolases).
The mean values of apparent activation energy were calculated using the linearized Arrhenius equation (fig. 6b, d). The values obtained for apparent activation energy (Ea) of APA were 53.8 and 48.6 kJ mol-1 for seawater and sediment, respectively. These values are similar to the activation energy of benthic fluxes of phosphate measured in situ in the Bay of Cádiz by Forja et al. (1994), who obtained a value of 45.0 kJ mol-1.
Influence of substrate concentration
]]> The kinetic parameters Km and Vmax were determined using the Lineweaver-Burk plot. The agreement between the experimental results and the model can be appreciated in figure 7. The values obtained for Km and Vmax were 10.6 mM and 89 µg of 4-nitrophenol g-1 h-1 and 8.42 mM and 145 µg of 4-nitrophenol L-1 h-1 for sediment and seawater, respectively.On the basis of these two results, the optimum analytical conditions for determining potential phosphatase activities in seawater and sediment in the study area were established. These conditions are summarised in table 1.
The optimum pH level for potential phosphatase activity varied depending on the sampling station and season. Nevertheless, in order to compare the results, the values selected were pH 5.5 and 8.5 for AcPA and APA, respectively.
The incubation time selected was 24 hours, because no significant increase in the number of microorganisms responsible for phosphatase activity was observed after this period (unpublished data). The incubation of samples with or without sodium azide showed no significant differences (P < 0.05). An incubation temperature of 28°C was selected as optimum.
The methodology established allows the quantification of the phosphatase activity derived from the particulate matter of the seawater in the salt-pond. However, the total amount of phosphatase activity in seawater is the sum of the activity from the dissolved fractions and the particulate matter; the proportion varies depending on sample origin and season. In general, phosphatase activity from the particulate matter represents 75-90% of the total phosphatase activity (unpublished results). A similar range for enzymatic activity associated with particulate matter was reported by Huber and Kidby (1984a) for the Peel Harvey Estuary and by Berman (1970) for Lake Kinneret. Berman et al. (1990) found that a high proportion of phosphatase activities was assigned to the specific size fraction of the natural microplankton (<0.8 µm >0.2 µm). Although bacteria and algae are producers of alkaline phosphatase, their relative importance depends on the season and is related to algal blooms (Berman et al., 1990). In the photic zone, the phosphatases are mainly of phytoplanktonic origin. Consequently, there is a high correlation between APA and chlorophyll a in seawater and lakes (Solorzano, 1978; Smith and Kalff, 1981; Stewart and Wetzel, 1982; Siuda, 1984; Huber and Kidby, 1984b). Similarly significant correlations were reported earlier between clorophyll a and APA and AcPA in the ecosystem under study (Frutos, 1996).
Spatial and temporal variations of phosphatase activity in the salt-pond
Figure 8 shows the profile of AcPA and APA at two sampling stations in the salt-pond. Station 1 is situated in the outer part of the pond and station 2 in the inner part, a zone with a relatively high degree of confinement. The rest of the sampling stations showed seasonal evolutions similar to that of station 2. Temperature values ranged from 6.5°C to 25°C in the summer. Salinity presented values below 40 at station 1, and between 30 and 50 at station 2, reaching values close to 80 in the inner part of the salt-pond. The phosphate concentrations were higher in the outer than inner sampling stations. In the first case, values higher than 10 µM were observed at the end of autumn, whereas values close to 8 µM were observed during the winter at the second station. In general, the lowest values were recorded in the salt-ponds with higher degree of confinement, because the input of HPO42- was low, and the limited renovation of water provoked an increase in phytoplankton and macrophyte biomass and in consequence a decrease in nutrient concentrations (Lubián et al., 1985). Chlorophyll a was higher at the inner station than at the outer station; this is because salt-ponds act as traps for organic matter and the regeneration processes generate increased primary productivity.
The high standard deviations observed are due to the wide variability of the two types of enzymatic activity in the seasonal samplings made at the same site. Other authors have also observed a similar variability in other littoral zones (Stevens and Parr, 1977; Siuda, 1984).
Temperature and phosphatase activities showed a positive relationship. With regard to phosphate concentration and phosphatase activities, a negative correlation was observed for APA and a positive correlation for AcPA. This behaviour indicates the different biochemical nature of both enzymatic activities.
In general, the phosphatase activity of both types measured in salt-ponds is higher than in other coastal areas. Huber et al. (1985) estimated an average annual value of 45 µg of 4-nitrophenol L-1 h-1 for the Peel and Harvey estuaries (SW Australia), and Taft et al. (1977) reported an average phosphatase activity of about 22 µg of 4-nitrophenol L-1 h-1 for Chesapeake Bay (USA). The values obtained for the Bay of Cádiz were two to three times higher than those observed for these other zones. Nevertheless, the comparison of results obtained with different methods and from different sources must be done cautiously, in part because they represent potential rates. The high potential rate of phosphorus regeneration reported agrees with the benthic flux measured in situ in the Bay of Cádiz by Forja et al. (1994), with an average value of 4.4 ± 2.5 mmol m-2 d-1. This value is higher than those found by other authors in littoral areas; e.g., 0.78 ± 0.05 mmol m-2 d-1 for the Potomac River estuary (USA) (Callender and Hammond, 1982). This is presumably a consequence of the high inputs of allocthonous organic matter in untreated urban sewage that the Bay of Cádiz receives. These inputs are discharged into an ecosystem with a reduced water volume and where seawater renewal by tidal action is limited (Blasco et al., 1987).
]]> The results obtained and the low values of the nutrient concentrations in the water of salt-ponds (Forja et al., 1990) suggest that in such systems the nutrient regeneration process plays an important role in phytoplankton production. Though there was considerable variability, it can be concluded that there are significant differences (P < 0.01) in phosphatase activity among the sampling stations. The highest phosphatase activity was observed in shallow sites and at greater distances from the point where seawater enters the salt-pond, with summer phosphatase levels at stations 2-5 greater than 100 µg of 4-nitrophenol L-1 h-1 during summer, while the annual average for both enzymatic activities is about 75 µg of 4-nitrophenol L-1 h-1. The high values presumably reflect that: (1) the input of phosphorus from outside is reduced and inorganic phosphorus exerts an inhibitory effect on phosphatase activity (Berman, 1970; Stevens and Parr, 1977; Kobori and Taga, 1979; Siuda, 1984; Chrost and Siuda, 1986); (2) in these highly confined areas, an increase in phytoplankton biomass and macrophytes is produced (Lubián et al., 1985), which in turn increases the enzymes in seawater, and these organisms are considered to be mainly responsible for the phosphatase activity in shallow systems (Solorzano, 1978; Smith and Kalff, 1981; Stewart and Wetzel, 1982; Siuda, 1984; Huber and Kidby, 1984b); and (3) high rates of insolation increase the summer water temperature to 25-40°C, the optimum range for enzymatic activities.Moreover, a marked, statistically significant (P < 0.05) seasonal evolution was observed in the inner part of the salt-pond during the sampling period. At most of the sampling stations in the inner part, the regenerative capacity of phosphorus was found to increase, since the circulation of seawater through the salt-pond is restricted from April to November. In this period, the values of phosphatase activity were 200% higher than when the circulation of seawater is not restricted. The increase reaches a maximum at the end of summer. Later, a decrease is observed and a minimum is observed in winter.
Potential phosphatase activity in the sediment
Sediment values are very high. The mean annual value in the salt-pond reached about 240 µg of 4-nitrophenol g-1 h-1 and 195 µg of 4-nitrophenol g-1 h-1 for APA and AcPA, respectively. These values are much higher than those found in other studies. For example, Sayler et al. (1979) estimated a mean value of 15 µg of 4-nitrophenol g-1 h-1 for the salt-ponds of Melton Hill in Tennesse, while Degobbis et al. (1986) recorded values of 10 µg of 4-nitrophenol g-1 h-1 for alkaline phosphatase activity in Venice Lagoon.
Sediments are influenced by numerous physical, chemical and biological factors, such as the location of the salt-pond, the degree of water renovation, granulometry and chemical composition of the sediment, and intensity of bioturbation activity. Therefore, it is difficult to establish a clear seasonal evolution. Also, the difficulty of obtaining representative samples is another factor that must be taken into account (Gómez-Parra and Frutos, 1987).
The vertical profiles of both types of enzymatic activity at sampling stations 1 and 4 are shown in figure 9. A similarity in the evolution of AcPA and APA in the sediment column can be observed. This is a consequence of both enzymatic activities having the same origin (Bhatti, 1978).
The evolution of the two types of enzymatic activity in the analysed core is very regular, the highest values being found in the surface sediment; when the depth increases, a decrease in the enzymatic activity is observed and at a depth of 15 cm, values close to zero were measured. Kobori and Taga (1979) found similar variation in the sediments of Tokyo Bay. These profiles are a result of the physicochemical characteristics of the water-sediment interface and the high nutrient fluxes across it (Forja et al., 1994). In consequence, a proliferation of microorganisms responsible for phosphatase activity, principally bacteria, occurs at the surface. In the sediments, the microorganism population diminishes in line with the depth (Krom and Berner, 1981; Degobbis et al., 1986).
It can be concluded that the analytical variables (pH of incubation medium, inoculate quantity, substrate concentration, temperature and incubation time) exert a notable influence on the determination of enzymatic activities. The importance of these variables varies among samples depending on their origin and on the season when they were collected.
]]> The average annual values for both types of enzymatic activity (AcPA and APA) were relatively high for both seawater and sediment. This is due to three factors: (1) the high input of allocthonous organic matter in the Bay of Cádiz, which is the substrate for enzymatic activities; (2) the climatic conditions of this area, particularly the high average annual temperatures; and (3) the high degree of confinement of the seawater in salt-ponds. The rate of the mineralization process of inorganic phosphorus shows that the salt-ponds of the Bay of Cádiz are very productive systems, where the regeneration process plays an essential role in supporting the high biomass of the salt-ponds.
Acknowledgements
We thank I. Fernández, A. Vidal and P. Vidal for their help in the collection and processing of the samples, and M. Hampel and R. Snart for revision of the English language manuscript.
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