Mierob Ecol (1987) 13:141-158
MICROBIAL ECOLOGY (~ Springer-VerlagNew York Inc. 1987
A Model for Acid and Alkaline Phosphatase Activity in a Small Pond Milan Matavulj* and Ken P. Flint Department of Environmental Sciences, University of Warwick, Coventry,England
Abstract. Acid and alkaline phosphatase activity were determined in a small pond over a period o f 24 months (64 samples). Activity o f each phosphatase enzyme was positively correlated with chlorophyll concentration, viable bacterial count, total phosphate concentration, inorganic phosphate concentration, and temperature. Multiple regression analysis was used to formulate equations that described phosphatase activity in terms o f these physical, chemical, and biotic factors. Corrected coefficients o f determination were calculated, and the highest values were obtained when all parameters were included in the equation (r 2 = 0.776 and 0.659 for alkaline and acid phosphatase activity, respectively). However, there was little improvement in the r 2 value obtained when only chlorophyll was used in the equation (r 2 = 0.654 and 0.624, respectively). Samples were then taken over a further 12 months (25 samples), and observed activity was compared with the activity predicted by application o f the previously derived equations. For alkaline phosphatase, the best fit between observed and expected activity was seen with the equation containing all parameters, but for acid phosphatase the best fit was seen with the equation containing only chlorophyll and temperature as the determinants. In both cases there was a good fit between observed and expected data using the equation containing chlorophyll as the sole determinant. From this we have concluded that phytoplankton were the chief producers of phosphatase activity in this pond, although the influence o f physical and chemical factors on enzyme activity could not be ignored. Introduction Over recent years, the possibility of cultural eutrophication destroying natural water communities has stimulated research into how algae develop in these water bodies, and the idea that a single nutrient could limit growth has been one operational approach [40]. The measurement o f nutrient levels in water samples, particularly at times of algal maxima, is often not enough to identify the growth-limiting nutrient for that water body. Three methods have been suggested to overcome this problem: (1) the enrichment of portions of water * Present address: Institute of Biology, PMF-Novi Sad, Dr llije Djuricica 6, 21000 Novi Sad, Yugoslavia.
142
M. Matavulj and K. P. Flint
with known nutrients, (2) chemical analyses of the algae and comparison with known nutrient-limited cells, and (3) the determination of some metabolic activity thal is specific to nutrient limitation and that changes quickly as the limitation changes. This last approach is based mainly on enzyme assays and, as phosphorus is considered to be the commonest growth limiting nutrient, phosphomonoesterases (phosphatases) have been considered indicators of phosphorus deficiency in aquatic environments [3, 32]. It must be remembered, however, that phosphorus alone does not control algal development, but is only one of a complex of chemical, physical, and biotic parameters which influence growth. Phosphatases are generally nonspecific enzymes that hydrolyze organic phosphates and some inorganic polyphosphates. The reaction can have an optimum pH of around 5.0 (acid phosphatases), 7.0 (neutral phosphatases), or greater than 9.0 (alkaline phosphatases). The properties of each of these enzymes are well documented for bacteria [5, 8, 11, 22, 25, 37, 39, M. Matavulj, K. P. Flint (1980) Proc. 2nd Intrnl. Symp. Microbial Ecology, p. 150 (Abstr.)], algae [1, 20, 21, 28], and fungi [18]. There have been various reports on phosphatase activities in both freshwater and marine environments, but most have dealt exclusively with alkaline phosphatase activity and the factors that influence this enzyme's activity [3, 9, 10, 15, 16, 32, 34]. Some studies have shown the presence of acid and alkaline enzymes; the relative proportion of each seems dependent upon the ambient pH, orthophosphate concentration, and microflora composition [9, 10, 34]. Phosphatase activity has been determined in the lakes of the English Lake District and a correlation has been shown to exist between the numbers of bacteria, alkaline phosphatase producers, total biomass, total phosphorus concentration, and alkaline phosphatase activity [ 14-16]. In Lake Kinneret, Israel, alkaline phosphatase activity was also correlated with dissolved total phosphate [3]. Alkaline phosphatase activity increased sharply after the peak of a Peridiniurn sp. bloom and was apparently related to inorganic phosphate limitation. Acid phosphatase activity remained relatively constant and was correlated with chlorophyll concentrations rather than orthophosphate concentrations. Both enzymes were predominantly cell or particle associated rather than extracellular, particularly during the bloom period [42]. There have also been some studies on marine environments, mainly in oligotrophic, open ocean regions [30]. However, an extensive study of alkaline phosphatase activity in a eutrophic marine environment, Tokyo Bay, revealed a positive correlation between log alkaline phosphatase activity and the log values for bacterial viable count, alkaline phosphatase producers, chlorophyll, protein, DNA, seston, inorganic phosphate concentration, and total phosphate concentration [36]. Usually the aim of these studies on aquatic environments has been described as an attempt to determine the influence of orthophosphate or total phosphate concentrations on phosphatase activity in the natural environment. Laboratory studies with microorganisms have shown that alkaline phosphatase is often a derepressible enzyme, synthesized only when orthophosphate is the limiting nutrient [ 18, 21, 37]. On the other hand, it has been shown that many aquatic bacteria produce constitutive phosphatase enzymes and that these enzymes
Phosphatase Activity in a Pond
143
w e r e d e t e c t a b l e e v e n i n o l i g o t r o p h i c e n v i r o n m e n t s [9]. It is n o t y e t p r o v e n t h a t p h o s p h a t a s e e n z y m e s a r e r e g u l a t e d i n t h e n a t u r a l e n v i r o n m e n t , as t h e y ar e i n the l a b o r a t o r y s t u d i e s , a l t h o u g h s o m e w o r k e r s h a v e f o u n d a n i n c r e a s e i n specific a l k a l i n e p h o s p h a t a s e a c t i v i t y w h e n p h o s p h a t e w a s l i m i t i n g in f r e s h w a t e r e n v i r o n m e n t s [3, 31, 32]. T h e r e h a v e b e e n a f e w a t t e m p t s t o m o d e l e n z y m e a c t i v i t i e s in f r e s h w a t e r e n v i r o n m e n t s [ 15, 38]. H o w e v e r , p h o s p h a t a s e s c o u l d b e a u s e f u l m o d e l b e c a u s e t h e y a r e e a s y to m e a s u r e a c c u r a t e l y a n d b e c a u s e t h e r e is m u c h i n f o r m a t i o n available f r o m laboratory studies on the regulation o f e n z y m e synthesis. In this s t u d y w e h a v e a t t e m p t e d to p r o d u c e e q u a t i o n s d e f i n i n g a c i d a n d a l k a l i n e p h o s p h a t a s e a c t i v i t y in a s m a l l m a n - m a d e p o n d o n t h e c a m p u s o f W a r w i c k U n i v e r s i t y . O b s e r v a t i o n s f r o m t w o y e a r s o f s t u d y w e r e u s e d to p r o duce these equations; these were then used to c o m p a r e o b s e r v e d and expected values o f p h o s p h a t a s e activity o v e r a further period o f study.
Materials and Methods
Sample Site and Sampling The sample site studied is a small, man-made pond (average depth 0.75 m) on the University of Warwick campus (OS ref. SP297 764). The pond has been in existence since 1973 and was chosen as the study site because it lacks a normal sediment. The bottom of the pond is lined with concrete, except where underwater plants grow in containers. This allowed us to study aquatic micoorganisms without the interference of the normal sediment microflora. Water samples were collected in l-liter sterile glass bottles from just under the surface. Upon collection the temperature of the surface water was recorded. All other assays were conducted on return to the laboratory. The site was sampled at least every two weeks from January 1979 to December 1981. The 1979-1980 data were used to derive the equations aimed at explaining phosphatase activity, and the 1981 data were used to test the similarity of observed phosphatase activity and that calculated using these equations.
Phosphatase Assay The basis for the assay of phosphomonoesterase activity in the water samples was the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate (pNPP disodium salt, Sigma) in buffer at 30"C. The reaction mixture contained 6.2 ml water, 0.7 ml sterile TTA buffer (0.33 M Tris, 0.33M TES (N-tris(hydroxymethyl)-2-aminoethane sulphonic acid, Sigma), 0.33 M acetic acid, pH adjusted to 5.0 with HC1 or 9.0 with NaOH), and 0.1 ml filter-sterile pNPP (5% w/v in sterile distilled water). The initial absorbance and the final absorbance, after the addition of 0.2 ml l0 M NaOH, were measured at 420 nm in 2-cm pathlength glass cuvettes on a Unicam SP600 spectrophotometer. The reaction mixture was usually incubated for 24 hours at 30"C. A control containing sterile distilled water in place of the water sample was included for each pH. Activity was expressed as uM pNP released per ml water sample per hour, and was the mean of duplicate samples. Total phosphatase activity was measured in untreated water samples and extracellular activity was determined in samples that had been filtered through a 0.45-# sterile Millipore Swinnex Filter. To minimize leakage of phosphatase activity by cell breakage, each filter was discarded after filtration of 25 ml of water. The pH/activity profile ofphosphatase activity was determined using TTA buffer (above), diluted 10-fold in sterile distilled water and adjusted to pH values of 4-11 at 0.5-unit intervals. The
144
M. Matavulj and K. P. Flint
cellular/particulate material was concentrated 100-fold by centrifugation and resuspended in sterile distilled water. Of this suspension 0.1 ml was added to 2.8 ml buffer, and 0.1 ml pNPP was added. The activity was recorded after incubation for up to 24 hours. All activities were expressed relative to that at pH 9.0.
Orthophosphate and Total Phosphate Assays Inorganic phosphate was recorded by the single solution method of Murphy and Riley [26]. The determination was made using 0.45-# Millipore-filtered water and was taken to represent dissolved (or soluble) inorganic phosphate, although it has been suggested that this often overestimates the true dissolved orthophosphate concentration due to the hydrolysis of labile organic phosphates and inorganic polyphosphates [31 ]. Samples were assayed in quadruplicate in acid-washed glassware and the absorbance at 880 nm recorded on a Unlearn SP600 spectrophotometer in 2-cm pathlength glass cuvettes. A blank of sterile, distilled, deionized water and a standard containing 1 nag PO4 literwere also included. Total dissolved phosphate was measured in 0.45-u Millipore-filtered water using the persulphateperchloric acid digestion method previously described [23].
Chlorophyll Determination Total chlorophyll was measured by the acetone extraction method previously described [ 17, 41]. Up to 1 liter of water was filtered through a Whatman GFC filter and the absorbance measured at 665 and 750 nm in 2-cm pathlength glass cuvettes after overnight extraction in acetone (90% v/v).
Viable Bacterial Counts Viable counts were determined on casitone-glycerol-yeast extract agar plates using the spread plate technique, after 6 days incubation at 25~ [33]. All dilutions were prepared in sterile distilled water.
Statistical Tests Spearman's rank correlation coefficient and x 2 test were carried out as described in Ref. 27. Multiple correlation analysis and multiple regression analysis were carried out using the Minster program on the University Burroughs B6700 computer. The values were compared for significance with those in standard tables [29].
Results
Seasonal Variation in Phosphatase Activity Figure 1 shows the changes in temperature (la), alkaline phosphatase activity (1 b), a c i d p h o s p h a t a s e a c t i v i t y (1 c), t o t a l p h o s p h a t e c o n c e n t r a t i o n (1 d), o r t h o p h o s p h a t e (I e), l o g v i a b l e b a c t e r i a l c o u n t (1 f), a n d c h l o r o p h y l l (1 g), d u r i n g t h e
Phosphatase Activity in a Pond
145
300 I
l~
(dl
o > -o
~
'IOC
. 9 L
. . . . . . . . . . . . .
I (bl
~30 m
MICROBIAL ECOLOGY (~ Springer-VerlagNew York Inc. 1987
A Model for Acid and Alkaline Phosphatase Activity in a Small Pond Milan Matavulj* and Ken P. Flint Department of Environmental Sciences, University of Warwick, Coventry,England
Abstract. Acid and alkaline phosphatase activity were determined in a small pond over a period o f 24 months (64 samples). Activity o f each phosphatase enzyme was positively correlated with chlorophyll concentration, viable bacterial count, total phosphate concentration, inorganic phosphate concentration, and temperature. Multiple regression analysis was used to formulate equations that described phosphatase activity in terms o f these physical, chemical, and biotic factors. Corrected coefficients o f determination were calculated, and the highest values were obtained when all parameters were included in the equation (r 2 = 0.776 and 0.659 for alkaline and acid phosphatase activity, respectively). However, there was little improvement in the r 2 value obtained when only chlorophyll was used in the equation (r 2 = 0.654 and 0.624, respectively). Samples were then taken over a further 12 months (25 samples), and observed activity was compared with the activity predicted by application o f the previously derived equations. For alkaline phosphatase, the best fit between observed and expected activity was seen with the equation containing all parameters, but for acid phosphatase the best fit was seen with the equation containing only chlorophyll and temperature as the determinants. In both cases there was a good fit between observed and expected data using the equation containing chlorophyll as the sole determinant. From this we have concluded that phytoplankton were the chief producers of phosphatase activity in this pond, although the influence o f physical and chemical factors on enzyme activity could not be ignored. Introduction Over recent years, the possibility of cultural eutrophication destroying natural water communities has stimulated research into how algae develop in these water bodies, and the idea that a single nutrient could limit growth has been one operational approach [40]. The measurement o f nutrient levels in water samples, particularly at times of algal maxima, is often not enough to identify the growth-limiting nutrient for that water body. Three methods have been suggested to overcome this problem: (1) the enrichment of portions of water * Present address: Institute of Biology, PMF-Novi Sad, Dr llije Djuricica 6, 21000 Novi Sad, Yugoslavia.
142
M. Matavulj and K. P. Flint
with known nutrients, (2) chemical analyses of the algae and comparison with known nutrient-limited cells, and (3) the determination of some metabolic activity thal is specific to nutrient limitation and that changes quickly as the limitation changes. This last approach is based mainly on enzyme assays and, as phosphorus is considered to be the commonest growth limiting nutrient, phosphomonoesterases (phosphatases) have been considered indicators of phosphorus deficiency in aquatic environments [3, 32]. It must be remembered, however, that phosphorus alone does not control algal development, but is only one of a complex of chemical, physical, and biotic parameters which influence growth. Phosphatases are generally nonspecific enzymes that hydrolyze organic phosphates and some inorganic polyphosphates. The reaction can have an optimum pH of around 5.0 (acid phosphatases), 7.0 (neutral phosphatases), or greater than 9.0 (alkaline phosphatases). The properties of each of these enzymes are well documented for bacteria [5, 8, 11, 22, 25, 37, 39, M. Matavulj, K. P. Flint (1980) Proc. 2nd Intrnl. Symp. Microbial Ecology, p. 150 (Abstr.)], algae [1, 20, 21, 28], and fungi [18]. There have been various reports on phosphatase activities in both freshwater and marine environments, but most have dealt exclusively with alkaline phosphatase activity and the factors that influence this enzyme's activity [3, 9, 10, 15, 16, 32, 34]. Some studies have shown the presence of acid and alkaline enzymes; the relative proportion of each seems dependent upon the ambient pH, orthophosphate concentration, and microflora composition [9, 10, 34]. Phosphatase activity has been determined in the lakes of the English Lake District and a correlation has been shown to exist between the numbers of bacteria, alkaline phosphatase producers, total biomass, total phosphorus concentration, and alkaline phosphatase activity [ 14-16]. In Lake Kinneret, Israel, alkaline phosphatase activity was also correlated with dissolved total phosphate [3]. Alkaline phosphatase activity increased sharply after the peak of a Peridiniurn sp. bloom and was apparently related to inorganic phosphate limitation. Acid phosphatase activity remained relatively constant and was correlated with chlorophyll concentrations rather than orthophosphate concentrations. Both enzymes were predominantly cell or particle associated rather than extracellular, particularly during the bloom period [42]. There have also been some studies on marine environments, mainly in oligotrophic, open ocean regions [30]. However, an extensive study of alkaline phosphatase activity in a eutrophic marine environment, Tokyo Bay, revealed a positive correlation between log alkaline phosphatase activity and the log values for bacterial viable count, alkaline phosphatase producers, chlorophyll, protein, DNA, seston, inorganic phosphate concentration, and total phosphate concentration [36]. Usually the aim of these studies on aquatic environments has been described as an attempt to determine the influence of orthophosphate or total phosphate concentrations on phosphatase activity in the natural environment. Laboratory studies with microorganisms have shown that alkaline phosphatase is often a derepressible enzyme, synthesized only when orthophosphate is the limiting nutrient [ 18, 21, 37]. On the other hand, it has been shown that many aquatic bacteria produce constitutive phosphatase enzymes and that these enzymes
Phosphatase Activity in a Pond
143
w e r e d e t e c t a b l e e v e n i n o l i g o t r o p h i c e n v i r o n m e n t s [9]. It is n o t y e t p r o v e n t h a t p h o s p h a t a s e e n z y m e s a r e r e g u l a t e d i n t h e n a t u r a l e n v i r o n m e n t , as t h e y ar e i n the l a b o r a t o r y s t u d i e s , a l t h o u g h s o m e w o r k e r s h a v e f o u n d a n i n c r e a s e i n specific a l k a l i n e p h o s p h a t a s e a c t i v i t y w h e n p h o s p h a t e w a s l i m i t i n g in f r e s h w a t e r e n v i r o n m e n t s [3, 31, 32]. T h e r e h a v e b e e n a f e w a t t e m p t s t o m o d e l e n z y m e a c t i v i t i e s in f r e s h w a t e r e n v i r o n m e n t s [ 15, 38]. H o w e v e r , p h o s p h a t a s e s c o u l d b e a u s e f u l m o d e l b e c a u s e t h e y a r e e a s y to m e a s u r e a c c u r a t e l y a n d b e c a u s e t h e r e is m u c h i n f o r m a t i o n available f r o m laboratory studies on the regulation o f e n z y m e synthesis. In this s t u d y w e h a v e a t t e m p t e d to p r o d u c e e q u a t i o n s d e f i n i n g a c i d a n d a l k a l i n e p h o s p h a t a s e a c t i v i t y in a s m a l l m a n - m a d e p o n d o n t h e c a m p u s o f W a r w i c k U n i v e r s i t y . O b s e r v a t i o n s f r o m t w o y e a r s o f s t u d y w e r e u s e d to p r o duce these equations; these were then used to c o m p a r e o b s e r v e d and expected values o f p h o s p h a t a s e activity o v e r a further period o f study.
Materials and Methods
Sample Site and Sampling The sample site studied is a small, man-made pond (average depth 0.75 m) on the University of Warwick campus (OS ref. SP297 764). The pond has been in existence since 1973 and was chosen as the study site because it lacks a normal sediment. The bottom of the pond is lined with concrete, except where underwater plants grow in containers. This allowed us to study aquatic micoorganisms without the interference of the normal sediment microflora. Water samples were collected in l-liter sterile glass bottles from just under the surface. Upon collection the temperature of the surface water was recorded. All other assays were conducted on return to the laboratory. The site was sampled at least every two weeks from January 1979 to December 1981. The 1979-1980 data were used to derive the equations aimed at explaining phosphatase activity, and the 1981 data were used to test the similarity of observed phosphatase activity and that calculated using these equations.
Phosphatase Assay The basis for the assay of phosphomonoesterase activity in the water samples was the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate (pNPP disodium salt, Sigma) in buffer at 30"C. The reaction mixture contained 6.2 ml water, 0.7 ml sterile TTA buffer (0.33 M Tris, 0.33M TES (N-tris(hydroxymethyl)-2-aminoethane sulphonic acid, Sigma), 0.33 M acetic acid, pH adjusted to 5.0 with HC1 or 9.0 with NaOH), and 0.1 ml filter-sterile pNPP (5% w/v in sterile distilled water). The initial absorbance and the final absorbance, after the addition of 0.2 ml l0 M NaOH, were measured at 420 nm in 2-cm pathlength glass cuvettes on a Unicam SP600 spectrophotometer. The reaction mixture was usually incubated for 24 hours at 30"C. A control containing sterile distilled water in place of the water sample was included for each pH. Activity was expressed as uM pNP released per ml water sample per hour, and was the mean of duplicate samples. Total phosphatase activity was measured in untreated water samples and extracellular activity was determined in samples that had been filtered through a 0.45-# sterile Millipore Swinnex Filter. To minimize leakage of phosphatase activity by cell breakage, each filter was discarded after filtration of 25 ml of water. The pH/activity profile ofphosphatase activity was determined using TTA buffer (above), diluted 10-fold in sterile distilled water and adjusted to pH values of 4-11 at 0.5-unit intervals. The
144
M. Matavulj and K. P. Flint
cellular/particulate material was concentrated 100-fold by centrifugation and resuspended in sterile distilled water. Of this suspension 0.1 ml was added to 2.8 ml buffer, and 0.1 ml pNPP was added. The activity was recorded after incubation for up to 24 hours. All activities were expressed relative to that at pH 9.0.
Orthophosphate and Total Phosphate Assays Inorganic phosphate was recorded by the single solution method of Murphy and Riley [26]. The determination was made using 0.45-# Millipore-filtered water and was taken to represent dissolved (or soluble) inorganic phosphate, although it has been suggested that this often overestimates the true dissolved orthophosphate concentration due to the hydrolysis of labile organic phosphates and inorganic polyphosphates [31 ]. Samples were assayed in quadruplicate in acid-washed glassware and the absorbance at 880 nm recorded on a Unlearn SP600 spectrophotometer in 2-cm pathlength glass cuvettes. A blank of sterile, distilled, deionized water and a standard containing 1 nag PO4 literwere also included. Total dissolved phosphate was measured in 0.45-u Millipore-filtered water using the persulphateperchloric acid digestion method previously described [23].
Chlorophyll Determination Total chlorophyll was measured by the acetone extraction method previously described [ 17, 41]. Up to 1 liter of water was filtered through a Whatman GFC filter and the absorbance measured at 665 and 750 nm in 2-cm pathlength glass cuvettes after overnight extraction in acetone (90% v/v).
Viable Bacterial Counts Viable counts were determined on casitone-glycerol-yeast extract agar plates using the spread plate technique, after 6 days incubation at 25~ [33]. All dilutions were prepared in sterile distilled water.
Statistical Tests Spearman's rank correlation coefficient and x 2 test were carried out as described in Ref. 27. Multiple correlation analysis and multiple regression analysis were carried out using the Minster program on the University Burroughs B6700 computer. The values were compared for significance with those in standard tables [29].
Results
Seasonal Variation in Phosphatase Activity Figure 1 shows the changes in temperature (la), alkaline phosphatase activity (1 b), a c i d p h o s p h a t a s e a c t i v i t y (1 c), t o t a l p h o s p h a t e c o n c e n t r a t i o n (1 d), o r t h o p h o s p h a t e (I e), l o g v i a b l e b a c t e r i a l c o u n t (1 f), a n d c h l o r o p h y l l (1 g), d u r i n g t h e
Phosphatase Activity in a Pond
145
300 I
l~
(dl
o > -o
~
'IOC
. 9 L
. . . . . . . . . . . . .
I (bl
~30 m
