The Alkaline Phosphatase Activity of Microplankton in

AASCIT Journal of Environment
2015; 1(2): 21-27
Published online June 10, 2015 (http://www.aascit.org/journal/environment)
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
1
Institut supérieur des professions infirmières et techniques de santé (ISPITS). AV. Abdelkhalek
Torres, Saniat Rmel, civil Hospital, Tetouan, Morocco
2
Faculty of science and technology (FST), Department of life sciences, Fes saiss, Morocco
3
Faculty of science Dhar El Mehrez, Department of biology, Fez, Morocco
Email address
Keywords
Reservoir,
Bacteria,
Algae,
Alkaline Phosphatase,
Phosphorus,
Seasonal Variations
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
Received: March 30, 2015
Revised: April 22, 2015
Accepted: April 23, 2015
Bacterial and phytoplanktonic alkaline phosphatase activity (APA) was measured from
March to December 1998 in the mesotrophic Allal El Fassi reservoir located in a semiarid 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-, HPO42-, 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-PO43–) 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
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-PO43- and PPT. 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
22
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-PO43-.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
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 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
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.h1
) 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
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
26
[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, 943948.
[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.