AASCIT Journal of Environment

2015; 1(2): 21-27 Published online June 10, 2015 (http://www.aascit.org/journal/environment)

Keywords Reservoir, Bacteria, Algae, Alkaline Phosphatase, Phosphorus, Seasonal Variations Received: March 30, 2015 Revised: April 22, 2015 Accepted: April 23, 2015

The Alkaline Phosphatase Activity of Microplankton in the Moroccan Mesotrophic Reservoir (Allal El Fassi)

Raoui S. M.1, Rachiq S.

2, Alaoui Mhamdi M.

3, Chadli N.

2

1Institut supérieur des professions infirmières et techniques de santé (ISPITS). AV. Abdelkhalek

Torres, Saniat Rmel, civil Hospital, Tetouan, Morocco 2Faculty of science and technology (FST), Department of life sciences, Fes saiss, Morocco 3Faculty of science Dhar El Mehrez, Department of biology, Fez, Morocco

Email address siraoui@yahoo.fr (Raoui S. M.), s.rachiq@voila.fr (Rachiq S.),

alaouimohammed@hotmail.com (Alaoui M. M.), nourchadli@hotmail.com (Chadli N.)

Citation Raoui S. M., Rachiq S., Alaoui Mhamdi M., Chadli N.. The Alkaline Phosphatase Activity of

Microplankton in the Moroccan Mesotrophic Reservoir (Allal El Fassi). AASCIT Journal of

Environment. Vol. 1, No. 2, 2015, pp. 21-27.

Abstract Bacterial and phytoplanktonic alkaline phosphatase activity (APA) was measured from March to December 1998 in the mesotrophic Allal El Fassi reservoir located in a semi-arid zone (Morocco). The total APA varied between 0,107-1,780 mmol PNP·L-1·h-1. In epilimnion, the bacteria contributed significantly (> 60%) to the total APA. In the meta and hypolimnion, the APA was predominantly algal (> 58%). The absence of correlation between APA and orthophosphates indicates that the hydrolysis caused by this enzyme was not a significant process in the recycling of phosphorus in Allal El Fassi reservoir. So, the APA was not a valid test of phosphorus deficiency.

1. Introduction

The dam Allal El Fassi is a Moroccan tank whose watershed is subject to intense agricultural activity. A major part of the phosphorus entering the tank, leaching the soil, has led to signs of precise eutrophication in this deduction (BOUHADDIOUI, 1997). On the other hand, it is accepted that phosphorus is the nutrient that contributes most to the process of eutrophication of aquatic ecosystems. (CAVALCANTE, 1994 ; VOLLEBWEIDER, 1969). However, the form of phosphorus, microplankton use preferentially is soluble and inorganic phosphorus H2PO4-, HPO4

2-, PO43- (CHROST and

OVERBECK, 1987). In condition of phosphorus deficiency, the planktonic species synthesize the alkaline phosphatase, an enzyme which hydrolyzes organophosphorus compounds and releases the soluble mineral phosphorus (GAGE and GORHAM, 1985; REICHARDT et al., 1967). Many authors have noted an inverse correlation between the APA and orthophosphate (BOAVIDA and MARK, 1995; CHROST et al., 1984 ; HEATH and COOKE, 1975), while in other aquatic ecosystems, these two variables are not proved (BOAVIDA and MARK, 1995; JAMET et al., 1997). The goal of this work is to study the part of microplankton in the APA and the relationship between this activity and the phosphorus (P-PT, P-PO4

3–) in Allal El Fassi reservoir.

2. Materials and Methods

Allal El Fassi dam, mesotrophic lake, is located in a semi-arid zone (34 ° N, 5 ° 40'W)

AASCIT Journal of Environment 2015; 1(2): 21-27 22

on the Oued Sebou 47 km from the city of Fez (figure 1) and was put in water in 1992. Its volume is 84.106 m3 and its maximum depth reaches 34 m. Bimonthly water withdrawals were made at the deepest point of the Lake, between 27th March and 21st December 1998, using a horizontal bottle Van Dorn type. The tested depths are 0 m, 2 m, 5 m, 10 m and near the bottom (bottom - 1 m). The abiotic parameters studied are temperature and concentrations of P-PO4

3- and P-PT. The latter are determined according to the standard methods reported by GOLTERMAN et al. (1978). Bacterial abundances are determined by epifluorescence microscopy according to the protocol proposed by HOBBIE and al. (1977), advocated as fluorochrome DAPI (WEAR and FEIG, 1980). The phytoplankton cells are counted using an inverted microscope (UTERMOHL, 1958), as amended by LEGENDRE and WATT (1971). Chlorophyll a was extracted with 90% acetone and dosed according to the technique reported by SCOR-UNESCO (1966). In order to remove

most living or non-living particles large, raw water is filtered throughout a 100 µm net of mesh vacuum. The determination of the APA is performed on samples of 100 mL (triplicatas), immediately filtered moderate vacuum (< 100 mm Hg) through 0,22 µm porosity nucleopore membranes and 0,65 µm. These filters placed in sterile test tubes are put in contact with a solution (3 mL) containing buffer (Tris-HCl 0,1 M, Mg++ 10-3 M, pH 8,5) and 1 mg.mL-1 of p-nitrophenyl phosphate (p-NPP). After incubation at 37 °C with slight agitation for 6 hours, the tubes are placed on ice to stop the reaction. Spectrophotometric water reading is performed at 410 nm (REICHARD and al., 1967). The results are expressed in mmol p-Nitrophenol (PNP) released per litre and per hour for three size fractions 0,22-100 µm (total APA) ; 0,65-100 µm (APA related to phytoplankton) and a fraction of 0,22-0,65 µm (APA related to bacteria). Free APA is performed on the fraction dissolved after filtration of the samples through a 0,22 µm porosity filter.

Figure 1. Geographic localisation of the Allal El Fassi reservoir.

3. Results

Allal El Fassi reservoir is a warm monomictic lake (extreme values: 10 to 29 °C) with a thermal stratification of the water which runs from the spring to the summer (figure 2). Orthophosphate concentrations range between 0 (several times to 0 m) and 0,2 mg P-PO4

3-.L-1 (July 15th near the bottom) (figure 3). Changes in total phosphorus follow those of orthophosphate and show the values that vary between 0,001 mg P-PT.L-1 (on several occasions at 0 m) and 0,69 mg P-PT.L-1 (September 9th near the bottom) (figure 3). Phytoplankton densities vary between 0,30.105 cells.L-1 (July 15th near the bottom) and 28.105 cells.L-1 (May 26th at 5 m). The average value throughout the study is 6,14.105 cells.L-1

(figure 4). The average concentrations of chlorophyll a range between 1,20 and 4,80 µg.L-1. During the spring and summer, phytoplankton was dominated by diatoms represented by Cyclotella ocellata. This species has been accompanied during the fall season by a population composed of Cryptomonas ovata, Peridinium cinctum, Dinobryon

sertularia. Bacterial abundance ranged from 1,01.106 Bact.ml-1 (August 24 near the bottom) to 9,86.106 Bact.ml-1 (April 17th to 10 m). The average of the total withdrawals of 5,34. 106 Bact.ml-1 (figure 5). These abundances are relatively homogeneous at levels 0 m, 2 m, 5 m and 10 m (5. 106 Bact.ml-1), whereas they decrease (3,6. 106 Bact.ml-1) at the level of the bottom of the lake.

23 Raoui S. M. et al.: The Alkaline Phosphatase Activity of Microplankton in the Moroccan Mesotrophic Reservoir (Allal El Fassi)

Figure 2. Seasonal variations of water temperature at different depths of water column.

Figure 3. Spatial-temporal variations of orthophosphates and total

phosphorus at different depths of water column.

Figure 4. Spatial-temporal variations of algal densities at different depths of

water column.

AASCIT Journal of Environment 2015; 1(2): 21-27 24

Figure 5. Spatial-temporal variations of bacterial abundances at different

depths of water column.

No APA related to the dissolved fraction could be measured regardless of reported depth. Total APA presents values that fluctuate between 0,107 mmol PNP.L-1.h-1 (September near the bottom) and 1,780 mmol PNP.L-1.h-1 (July 29th at 0 m) (figure 6). Strong activities are recorded during thermal stratification in summer. Statistical analysis by linear regression leaves a significant correlation between the APA and the temperature (0,53 < r < 0,72 ; p < 0,05) at 0 m, 2 m and 5 m levels. However, this activity does not have significant correlation (p > 0,05) with concentrations of

orthophosphate and total phosphorus, along the water column. The relative contribution of the fraction 0,22-0,65 µm size is high (> 60% of total APA) 0 m and 2 m (figure 7). At these levels, the APA of this fraction is significantly correlated with bacterial abundance (0,64 < r < 0,75 ; p < 0,01). On the other hand, the strong APA related to the total fraction 0,65-100 µm (> 58% of APA) is recorded at 5 m and 10 m (figure 7). At these depths, this APA is bound by a significant correlation to the algal density (0,74 < r < 0,80 ; p < 0,05) and chlorophyll a (0,56 < r < 0,62 ; p < 0,05). These positive correlations undoubtly demonstrate a significant dependency between the APA and microplankton (bacteria, algae) in this reservoir. However, we note the absence of significant correlation between the two APA microplanktonic fractions and concentrations of orthophosphate and total phosphorus along the water column.

Figure 6. Spatial-temporal variations of total APA at different depths of

water column.

25 Raoui S. M. et al.: The Alkaline Phosphatase Activity of Microplankton in the Moroccan Mesotrophic Reservoir (Allal El Fassi)

Figure 7. Relative contribution of APA in two size fractions of microplankton.

4. Discussion

The APA is often considered as an indication of deficiency of phosphorus in aquatic area (PETTERSON, 1980 ; GAGE and GORHAM, 1985). These authors showed the existence of significant inverse correlation between the APA and inorganic phosphorus concentrations. During this study, we note the absence of significant correlation (p > 0,05) between the APA and orthophosphate concentrations in all fractions of size and at all depths tested. Our results suggest an independence between the APA and the phosphorus, at least during the period of the study. Similar results were also observed by JAMET and al. (1995, 1997). The same way, BOAVIDA and MARK (1995) have found the existence of a positive correlation between the APA and the concentrations of phosphomonoesters reflecting the fact that they do not represent a substrate for this enzyme. Furthermore,

JANSSON and al. (1988) showed that the strong APA are recorded when phosphorus concentrations are extremely low. At the level of the superficial layers, the fall of orthophosphate may be probably related to assimilation by bacteria or algae (CAPBLANCQ, 1990) or their complexation with dissolved oxygen (ALAOUI and al., 1994).

As soon as we approach the deep layers, the total phosphorus and orthophosphate concentrations gradually increase and reach their maximum at the bottom level. If our results indicate that hydrolysis by the APA is not a significant mechanism in recycling of phosphorus, there is probably other phosphorus sources, including the external inputs by the Sebou (BOUHADDIOUI, 1997), autolysis of bacteria and senescence of algal (MONTIGUY and PRAIRIE, 1993) cells and the process of leaching of this element from the sediment (ALAOUI and al,. 1994). Total APA registered for this tank (0,107 to 1,78 mmol.PNP.L-1.h-

1) exceeds those reported in different temperate Lakes (BOAVIDA and MARK, 1995; KALINOWSKA, 1997; JAMET and al., 1997). The highest values of this activity were recorded at the level of the surface layers in the summer at thermal stratification. This increase may be related to the environmental conditions of the environment. Indeed, the high temperature throughout the year, including in the epi and the metalimnion, probably promotes an increase in the synthesis of this enzyme. But at the bottom level, the low values of the APA would be related, on the one hand, to the downward temperature and, on the other hand, to low bacterial densities and the physiological state of the senescent phytoplankton cells. According to the results recorded by FRANCKO (1983) and those of JAMET and al. (1997), the total APA is fully associated with bacteria and algae insofar as no trace of this enzyme activity was revealed in the dissolved fraction. This lack of free APA could be related to trophic level of the environment. The absence of APA in the dissolved fraction is registered also in the mesotrophic Lake Pavin (JAMET and al., 1997), while it is generally important in eutrophic and hypereutrophic environments (JAMET, 1995; KALINOWSKA, 1997). Depending on the depth, total APA sometimes presents bacterial and sometimes phytoplankton dominance. At the level of the superficial layers, the size fraction 0,22-0,65 µm, bacterial majority, represents the major part of the total APA, while it decreased with depth for the benefit of that associated with the fraction of size 0,65-100 µm to phytoplankton dominance. This distribution is in relation to spatiotemporal variations of bacterioplankton whose maximum abundance is registered on the surface of the restraint. The strong total algal contribution to the APA could be attributed to the appearance in this reservoir of algal population dominated largely by the diatomophycees represented by C. ocellata. HINO (1988) and OLSSON (1990) it showed that phytoplankton biomass contributes significantly to the APA when it is dominated by diatoms and cyanobacteria.

AASCIT Journal of Environment 2015; 1(2): 21-27 26

5. Conclusion

The total APA measured in Allal El Fassi reservoir is fully associated with bacterial and algal communities. The importance of the relative contribution of bacterial and algal communities in the APA is a function of depth. During the thermal stratification, the bacterioplankton represented the major part of the total APA, while it decreased with depth for the benefit of that associated with the phytoplankton. The lack of significant correlation between the APA and orthophosphate concentrations suggests that alkaline phosphatase is not be a reliable indicator of orthophosphate deficiency.

Acknowledgement

Our thanks to regional hydraulics service of Fez and the staff of the Allal El Fassi dam.

References

[1] ALAOUI MHAMDI M., ALEYA L., RACHIQ S., DEVAUX J., 1994. Etude préliminaire sur les échanges de phosphore à l’interface eau-sédiment au sein de la retenue d’Al Massira (Maroc). Rev. Sci. Eau, 7, 115-130.

[2] BOAVIDA M.J., HEATH R.T., 1988. Is alkaline phosphatase always important in phosphate regeneration? Arch. Hdrobiol., 111, 507-5018.

[3] BOAVIDA M.J., MARQUE R.T., 1995. Low activity of alkaline phosphatase in tow eutrophic reservoir. Hydrobiologia, 297, 11-16.

[4] BOUHADDIOUI A., 1997. Bilan biogéochimique de l’azote et du phosphore, et dynamique des populations phytoplanctoniques de la retenue de barrage Allal El Fassi. Th. DES. FST, Fès saiss, 188p.

[5] CAPBLANCQ J., 1990. Nutrient dynamics and pelagic food web interaction in oligotrophic and eutrophic environments: an overview. Hydrobiologia, 207, 1-14.

[6] CAVALCANTE P.R., 1994. Etude sur la mobilisation du phosphore, des formes azotées et quelques métaux associés dans la vase eutrophe : expérimentation sur les vases de l’Erdre (in situ) et en laboratoire. Th. Doct. Univ., Nante, France, 164p.

[7] CHROST R.J., SIUDA W., HALEMEJKO G.Z., 1984. Longterm studies on alkaline phosphatase activity (APA) in lake with fish-aquaculture in relation to lake eutrophication and phosphorus cycle. Arch. Hydrobiol. Suppl., 70, 1-32.

[8] CHROST R.J., OVERBECK J., 1987. Kinetic of alkaline phosphatase activity and phosphorus availability for phytoplankton and bacterioplankton in lake Plub see (North German eutrophic lake). Microb. Ecol., 13, 229-248.

[9] FRANCKO D.A., 1983. Size-fraction of alkaline phosphatase activity in lake water by membrane filtration. J. Freshwat Ecol., 2, 305-309

[10] GAGE M.A., GORHAM E., 1985. Alkaline phosphatase activity and cellular phosphorus as an index of the phosphorus status of phytoplankton in Minnesota lake. Freshwat. Biol., 15, 227-233

[11] GOLTERMAN H.L., CLYMO R.S., OHNSTAD M.A.M., 1978. Methods for physical and chemical analyses of freshwater. IBP. Manuel n° 8, (2nd édition). Blanck. Sci. Publ., Oxford, 213 p.

[12] HEATH R.T., COOKE G.D., 1975. The significance of alkaline phosphatase in eutrophic lake. Int. Ver. Theo. Limnol. Verh., 19, 959-965.

[13] HINO S., 1988. Fluctuation of algal alkaline phosphatase activity and the possible mechanisms of hydrolysis of dissolved organic phosphorus in lake Barato. Hydrobiologia, 157, 77-84.

[14] HOBBIE J.E., DALAY R.J., JASPER S., 1977. Use of nucleopore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol., 33, 1225-1228.

[15] JAMET D., 1995. Variations temporelles de l'activité de la phosphatase alkaline associée à différentes classes de taille du matériel particulaire au sein de trois lacs du massif central de degré trophiques différents. Th. Doct. Univ., Blaise Pascal, France, 180 p.

[16] JAMET D., ALEYA L., DEVAUX. J., 1995. Diel changes in the alkaline phosphatase activity of bacteria and phytoplankton in the hypereutrophic Villerest reservoir (Roanne, France). Hydrobiologia, 300-301, 49-56.

[17] JAMET D., AMBLARD C., DEVAUX J., 1997. Seasonal changes in alkaline phosphatase activity of bacteria and microalgea in lake Pavin (Massif Central, France). Hydrobiologia, 347, 185-195.

[18] JANSSON M., OLSSON H., PETTERSSON K., 1988. Phosphatases: Origin, characteristics and functions in lakes.. Hydrobiologia, 170, 157-175.

[19] KALINOWSKA K., 1997. Eutrophication processes in a shallow, macrophyte dominated lake alkaline phosphatase activity in lake Luknajno (Poland). Hydrobiologia, 342/343, 395-399.

[20] LEGENDRE L., WATT W. D., 1971. On a rapid technique for plankton enumeration. Ann. Inst. Ocenogr., Paris, 58, 173-177.

[21] MONTIGUY C., PRAIRIE Y.T, 1993. The relative importance of biological and chemical processes in the release of phosphorus from a higly organic sediment. Hydrobio/ogia, 253, 141-150.

[22] OLSSON H., 1990. Phosphatase activity in relation to phytoplankton composition and pH in Swedish lake. Freshwat. Biol., 23, 353-362.

[23] PETTERSON K., 1980. Alkaline phosphatase activity and algal surplus phosphorus and phosphorus deficiency indicator in lake Erken. Arch. Hydrobiol., 89, 54-87.

[24] PORTER K.G., FEIG Y.S., 1980. Use of DAPI for identifying and counting aquatic microflore. Limnol. Oceanogr., 25, 943-948.

[25] REICHARDT W., OVERBECK J., STEUBING L., 1967. Free dissolved enzyme in lake waters. Nature, 216, 1345-1347.

[26] SCOR-UNESCO., 1966. Determination of phytoplankton pigments in sea water. SCOR-UNESCO., 69 p.

[27] UTERMÖHL H., 1958. Zur vervollkommung der quantitative phytoplankton methodik. Mitt. Int. Ver. Limnol., 9, 1-38.

27 Raoui S. M. et al.: The Alkaline Phosphatase Activity of Microplankton in the Moroccan Mesotrophic Reservoir (Allal El Fassi)

[28] VOLLENWEINDER R.A., 1969. A manual of methods measuring primary production in aquatic envirenment. IBP., Handbook 12, Blak. Sci. Publ., Oxford, 244 p.