contributions of phosphatase and microbial activity to internal phosphorus loading and their...

8/21/2019 Contributions of Phosphatase and Microbial Activity to Internal Phosphorus Loading and Their Relation to Lake Eutr

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102 Science in China: Series D Earth Sciences2006 Vol.49 Supp. I 102113

www.scichina.com www.springerlink.com

DOI: 10.1007/s11430-006-8110-z

Contributions of phosphatase and microbial activity to

internal phosphorus loading and their relation to

lake eutrophication

SONG Chunlei1,2, CAO Xiuyun1,2, LI Jianqiu1,LI Qingman1,CHEN Guoyuan1,2

& ZHOU Yiyong

1

1. Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China;

2. Graduate School of the Chinese Academy of Sciences, Beijing 100039, China

Correspondence should be addressed to Zhou Yiyong (email: [email protected])

Received September 16, 2005; accepted February 6, 2006

Abstract Phosphatase may accelerate the process of lake eutrophication through improving

phosphorus bioavailability. This mechanism was studied in three Chinese eutrophic shallow lakes

(Lake Taihu, Lake Longyang and Lake Lianhua). Phosphatase activity was related to the concentra-

tion of soluble reactive phosphorus (SRP) and chlorophyll a. Stability of dissolved phosphatase in

reverse micelles may be attributed to molecular size, conformation and active residues of the enzyme.At the site with Microcystisbloomed in Lake Taihu, dissolved phosphatase activity was higher and

more stable in micelles, SRP concentrations were lower in interstitial water, the contents of different

forms of phosphorus and the amounts of aerobic bacteria were lower while respiration efficiency was

higher in sediments. Phosphobacteria, both inorganic and organic and other microorganisms were

abundant in surface water but rare in sediments. Therefore, internal phosphorus may substantially flux

into water column by enzymatic hydrolysis and anaerobic release, together with mobility of bacteria,

thereby initiating the bloom. In short, biological mechanism may act in concert with physical and

chemical factors to drive the internal phosphorus release and accelerate lake eutrophication.

Keywords: dissolved phosphatase, reverse micelle, internal loading, phosphorus form of sediment, mi-

crobiology, eutrophication, Microcystisbloom.

1 Introduction

Eutrophication is one of the serious environmental

problems in the world with the symptom of excess

algal growth caused by the increase of nutrient input,

especially phosphorus. Internal phosphorus loading

was closely related to eutrophication, which may im-

pede the improvement of water quality even after de-

creasing the external loading[1]

. Alkaline phosphatasecan catalyze organic phosphorus into orthophosphate

in water body and sediments. This is an important ap-

proach for the supply of bioavailable phosphorus[2].

There must be a relation between alkaline phosphatase

and eutrophication[3]. Dissolved alkaline phosphatase

(DAP) may effectively accelerate the phosphorus cy-

cling due to the high proportion and continuity of

catalytic capability[4,5]. Based on the regulation of al-

kaline phosphatase synthesis, its secretion and stability,Li et al. proposed the use of alkaline phosphatase ac-


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Contributions of phosphatase and microbial activity to internal phosphorus loading 103

tivities for an assessment of the P-status[6]. However,

the relationship between DAP and internal phosphorus

loading as well as eutrophication has not been ad-dressed. Microorganisms may regulate the abiotic ef-

fect in the process of phosphorus exchange of sedi-

ment-water interface due to the fluctuating redox con-

ditions in the sediment[7]. In addition, the microbial

community structure likely plays a major and direct

role in the release and uptake of phosphorus from the

sediment[8]. The decrease in potential for P release

from exposed sediments was caused by a shift in bac-

terial community structure[9]. Microorganisms can de-

compose organic phosphorus, store the polyphos-phate[10], and lower the redox potential by consumingoxygen, thus enhancing the release of phosphorus

from the iron-P complexes[11]. Microorganisms play an

important role in the process of phosphorus cycling.

Lactate bacteria can resolve phosphate and make the

SRP concentration increase in the Dianchi Lake. The

enrichment of phosphorus caused by assemble phos-

phorus bacteria and the mineralization of organic

phosphorus after their death are two important ways

resulting in hydrated phosphate deposit in the lake

[12]

.In addition, protease activity was closely related to

nitrogen mineralization and release. Phosphatase ac-

tivity was related to phosphorus decomposition in the

sediment of West Lake[13] and polluted degree indi-

cated by microbial characteristics in the sediment of

Victoria Bay of Hongkong[14]. Hence, the study of ac-tivity and stability of dissolved phosphatase in benthic

layer of lakes, the characteristics of microbial commu-

nity, and phosphorus form of sediment would be help-

ful for understanding the mechanism of internal load-

ing cycling and the contribution of microbial commu-

nity to internal phosphorus loading in lakes.

DAP plays a crucial role in phosphorus cycling of

lakes. Its behavior varied in different lakes. In poly-

humic Lake Skjervatjern and Lake Mekkojarvi of

Germany, DAP accounted for 60%80% in total ac-

tivity[15]. It exhibited very low activity in turbid Aus-

tralian river[16]. In addition, the relation between DAP

and phosphorus is very complex. In oligotrophic Lake

Herrensee, DAP was responsible for more than half of

the total hydrolysis of organic phosphorus[17], butshowed relatively low activity in Lake Lawrence with

the lack of phosphorus, USA[18]. The relative propor-

tion of DAP was independent of phosphorus in Ger-

man Lake Schohsee[19]. It may represent either a major

or an insignificant proportion of total phosphatase infive Oklahoma lakes, depending on the limnological

characteristics of lake ecosystem[20]. Both zooplank-

ton[21]and bacteria[22]could support the growth of al-

gae by actively excreting dissolved phosphatase to

supply the bioavailable phosphorus in water, and ex-

tracellular substance produced by algae can provide

carbon source for bacteria. In short, dissolved phos-

phatase in lakes was diverse, which reflected the abil-

ity of plankton to adapt environments. The diversity of

DAP was shown in terms of its origin, catalytic effi-ciency and temporal and spatial variations, which re-fracted the complex ecological relations in lakes. Fur-

thermore, the cationic surfactants enhanced the acid

phosphatase activity, while anionic surfactants inhib-

ited the activity completely, and neutral surfactants

decreased the activity also, but had little or no effect

on kinetics of the enzyme. In unconventional medium,

both VmaxandKmvalues of phosphatase increased sig-

nificantly due to the slight folding of enzyme mole-

cule

[23]

. If we introduced unconventional mediummethod into the research of freshwater ecology,

namely adding surfactants and organic solvents with

different proportions to lake water filtered through

0.45-m filters with higher DAP activity, we may give

further description of DAP and its stability in this re-

verse micelle system.The reverse micelle is a transparent and thermody-

namic stability multi-phase organic system, which

consists of an aqueous micro-domain (water pool)

facing the polar heads of the surfactant that surrounds

this core interacting with the organic solvent, through

hydrophobic chains[24]. The water structure in the

aqueous core may resemble that of the water adjacent

to biological membranes, suggesting that this system

is a model of biological structure[25]. Thus, the enzy-

matic activity in the water pool of reverse micelle is

stable and may avoid the disturbance of microorgan-

isms.

This paper discussed the relationship between

phosphatase (especially activity and stability of dis-

solved phosphatase) and eutrophication in lakes withdifferent trophic levels (Lake Lianhua, Lake Longyangand Lake Taihu) at spatial and vertical scales. The ob-


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104 Science in China:Series D Earth Sciences

jectives of this study are to test the feasibility of ap-

plying micellar enzymology method to the research of

dissolved phosphatase in lakes and understand thecontribution of activity and stability of phosphatase as

well as microbial activity to internal loading and lake

eutrophication.

2 Materials and method

2.1 Lake situations and sampling sites

Lake Lianhua and Lake Longyang are situated in

Hanyang, Wuhan, China. Lake Longyang was a beau-

tiful shallow lake in the 1950s, and the area decreasedgradually due to increased wastewater discharges,

aquaculture and agriculture activities in the 1970s,

then reduced to 2/3 of the origin in the 1990s and lake

water was polluted severely. Lake Lianhua is a small

recreational lake, and water quality was improved

markedly due to the growth of submerged macro-

phytes. Lake Taihu is the third largest freshwater lake

in China, situated in the south of the Yangtze River

delta. Over the recent two decades, the lake has had a

deterioration of its water quality in many parts due to

increased anthropogenic inputs, especially in Lake

Wulihu, a large embayment of Lake Taihu, which is

relatively close with slow flowing velocity and long

water exchange period. The total area of Lake Wulihu

is 5.6 km2, with a length of 0.31.5 km (N-S), a

width of 6 km (E-W), and an average depth of about

1.95 m. Lake Wulihu is the most eutrophic area of

Lake Taihu. Therefore, dredging was started on a large

scale in 2003

2004. The basic data of studied lakesare shown in Table 1. In our investigation, two sam-

pling sites were designed respectively in Lake Lianhua

and Lake Longyang, and six sampling sites were cho-

sen in field experiment in Lake Wulihu, including

Sites A(313224.6N 1201322.6E), C(31321.7N

1201318.7E), F(313158.1N 1201323.0E) and

E located in Lake Wulihu, and Sites B and D

(313132.3N 1201237.6E) located in Lake Taihu.

Sites A and F were dredged before January 2004, Site

C was dredged after January 2004, Sites B, D and Ewere not dredged, and Site B Microcystis bloomed.

Sampling sites designs are shown in Fig. 1.

Table 1 Basic data of water quality in Lake Lianhua, Lake Longyangand Lake Taihu (Samples of Lake Lianhua were collected on April 16,

2004; those of Lake Longyang were collected on May 10, 2004)

Sampling sitesArea(km2)

Depth(cm)

Transparency(cm)

pH

Lake Lianhua 0.076 100 42 7.78

Lake Longyang 1.800 140 31 8.02Lake Wulihu (LakeTaihu)

5.600* 195* 50** 7.62*

* Data from ref. [26]; ** data from ref. [27].

Fig. 1. Sampling sites design and location of Lakes Lianhua, Long-yang and Taihu.

2.2 Samples collection and preparation

Surface water of Lake Lianhua and Lake Longyang

was collected on April 16 and May 10, 2004, respec-

tively. Sediments were collected either by a Peterson

grab sampler, or by a hand-driven stainless steel corer

50 cm long with an internal diameter of 5.4 cm inLake Taihu on January 13 and September 27, 2004.

Interstitial water was extracted from the sediment by


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centrifugation at 3000 r/m for 30 minutes. The con-

centrations of soluble reactive phosphorus (SRP) and

chlorophyll ain surface water were measured after all

water samples were filtered through pre-washed

0.45-m filters[28]. Filtered water sample was concen-

trated to 40 mL with a rotary evaporator at 35 for

analysis of the DAP in the reverse micelles. All sam-

ples were stored in a refrigeratory.

2.3 Size-fractionation of alkaline phosphatase activ-

ity (APA)

All water samples were filtered through 0.45 and

3.0 m membrane filters. The contributions of APA to

the algal and bacterial fractions were calculated asfollows:

A = U F (3.0) and B = F (3.0) F (0.45),

where A = activity in algal fraction, i.e. in fraction

larger than 3.0 m, B = activity in bacterial fraction,

i.e.in fraction 0.453.0 m, U = activity of unfiltered

water sample, i.e.total APA, F (3.0) = activity in water

sample prefiltered through 3.0 m, and F (0.45) = ac-

tivity in water sample filtered through 0.45 m. The

final p-nitro-phenylphosphate (pNPP) concentration

(0.3 mmol L1) was used for the size-fractionation of

APA[29].

2.4 DAP in reverse micelles

Each concentrated sample of 4.15 mL was injected

into a reverse micelle of 0.2 mol/L hexadecyltrime-

thylammonium bromide (CTAB) and 1 mol/L 1-

bytanol (as co-surfactant) in cyclehexane, and then

made this system clear by acute oscillation. The polar

cores of the micelles have the ability to solubilize asignificant amount of water and form the water pool.

The enzyme and substrate (pNPP) can interact with

each other in the water pool. The pH and water con-

tent W0was adjusted by 0.1 mol/L TrisHCl buffer[30].

The reverse micelles with concentrated water samples

of Lake Lianhua and Lake Longyang were incubated

in different W0conditions (pH 8.5, 37), different pH

conditions (W0 14, 37) and different temperature

conditions (pH 8.5, W014). The reverse micelles with

concentrated water samples of Sites B and D in LakeTaihu were incubated in W0 14, pH 8.5, temperature

37 conditions . The samples in the reverse micelles

were collected in different incubation time. The reac-

tion was started by injection of the pNPP dissolved in

0.1 mol/L Tris-HCl buffer. The alkaline phosphataseactivity was determined by monitoring the increase in

absorbance at 400 nm after 4 h at 37 . T he molar

extinction coefficient in reverse micelles was 11300

mol/Lcm1. The concentration of the substrate with

the aqueous core of reverse micelles was constant at

18 mmol/L in all experiments mentioned above[31].

2.5 Enumeration of different microbial functional

groups

Bacteria were enumerated using plate count tech-niques for different functional groups. The numbers of

aerobic bacteria, organic and inorganic phosphobacte-

ria of surface, overlying water and sediment in Sites A,

C and D of Lake Taihu were determined by the serial

10-fold dilutions plate method. The culture medium

for aerobic bacteria: beef extract 3 g, peptone 5g, NaCl

5 g, agar 1518 g, distilled water 1000 mL. The cul-

ture medium for organic phosphobacteria: D-glucose

10g, (NH4)2SO4 0.5g, MgSO4 0.3g, NaCl 0.3g, KCl

0.3g, FeSO4 0.036g, MnSO4 0.03g, CaCO3 5g, agar1518g, distilled water 1000 mL. There needed 1mL

fresh vitellus as organic phosphorus source per 1215

mL culture medium; The culture medium for inorganic

phosphobacteria: D-glucose 10g, (NH4)2SO4 0.5g,

MgSO4 0.3g, NaCl 0.3g, KCl 0.3g, FeSO4 0.036g,

MnSO4 0.03g, Ca3(PO4)2 2g, agar 1518g, distilled

water 1000 mL. Two replicate drops of 1 mL of three

dilutions sample were pipetted onto the plates. Pouring

plates started after cooling molten appropriate agar

medium for analysis of above three bacteria groups indarkness to a temperature of 48 . Then dilution sa m-

ples and agar medium were mixed thoroughly and al-

lowed to cool in darkness for 2030 min. All plates

were incubated at 28 for 72 h. Colonies were

counted and the colony forming unit (CFU) ml/L or

g/L was calculated. Respiration efficiency of sediment

was determined by alkali absorbing method[32].

2.6 Phosphorus form of sediment

Sediment phosphorus fractionation was carried outaccording to Golterman, which grouped sediment P

into iron-bound P (Fe(OOH)P), calcium-bound P


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phorus forms in surface sediment at the undredged site

(Site D) and SRP concentration in the interstitial water

(Sites B and D) were significantly higher than those at

the dredged sites (Sites A and C, Fig. 3; Table 2).

Chlorophyll a concentration showed the same trend

(Table 3). In addition, Microcystissp. bloom occurred

at Site B then.

(CaCO3P), acid soluble organic P (ASOP) and hot

NaOH extractable residual organic P (NaOHPextr)[33].

3 Results

3.1 Internal phosphorus loading and productivity

The concentrations of different phosphorus forms indifferent depths of sediment at the undredged sites(Sites E and C) were markedly higher than those at thedredged sites (Sites A and F, Fig. 2) in January 2004.In September, the concentrations of different phos-

3.2 Alkaline phosphatase activity and stability

Different size-fractionated APA in the interstitial

water was higher than those in the overlying water of

Fig. 2. The concentration of different form phosphorus at different depth of Lake Taihu (2004-01).

Table 2 The concentration of SRP in overlying and interstitial water of Lake Taihu (2004-09)

Sampling sites A B C DOverlying

waterInterstitial

waterOverlying

waterInterstitial

waterOverlying

waterInterstitial

waterOverlying

waterInterstitial

waterSRP (mgL1)0.018 0.019 0.040 0.022 0.024 0.017 0.022 0.126

Table 3 The concentration of chlorophyll ain surface water of Lake Taihu (2004-09)

Sampling sites A B C D

Surface water Overlyingwater

Surfacewater

Overlyingwater

Surfacewater

Overlyingwater

Surfacewater

OverlyingwaterChlorophylla(gL1)

0.056 32.55 12.68 1.67 11.72 13.02

, Undetectable.


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Fig. 3. The concentration of different form phosphorus at surface sedi-ment of Lake Taihu (2004-09).

different sampling sites of Lake Taihu. Horizontally,

the APA of Site B where Microcystis sp. bloomed

showed the higher level, especially the dissolved parts

(Fig. 4).

The activity of DAP in the interstitial water of Site

B in reverse micelles began to decrease after 144 h

incubation, whereas the responding value was 72 h in

the surface and overlying water (Fig. 5). The activityof DAP of Lake Longyang in micelles apparently ex-

hibited higher values than that in Lake Lianhua. The

latter decreased sharply at 48 h, and then dropped at

96 h again, whereas the former may retain relatively

high level for 192 h in different W0 conditions (Fig.

6(a)). DAP of Lakes Longyang and Lianhua in mi-

celles was unstable at higher temperature. At 50, the

activity began to decrease after 35 h and 65 h incuba-

tion, respectively. At 20 and 37, the activity of

DAP of Lake Lianhua (65 h) began to decrease prior

to Lake Longyang (170 h) (Fig. 6(b)); In different pH

conditions, the activity of DAP of Lake Longyang in

micelles appeared to decline after 170 h incubation,

whereas the responding value in Lake Lianhua was 40

h (Fig. 6(c)). Accordingly, the stability of DAP in mi-

celles was not affected by pH but closely related to itsorigin. Shortly, the eutrophic Lake Longyang showed

higher enzymatic activity and stability in micelles.

3.3 Bacterial abundance and respiration efficiency

Site D in Lake Taihu showed the highest abundance

of different functional bacterial groups in the surface,

overlying water and sediments among all sampling

sites (Table 4). In Site B, the bacterial abundances ex-

hibited relatively higher level in the surface water,

while aerobic bacterial abundance in sediment withhigher respiration efficiency was undetectable. Or-

ganic phosphobacteria and inorganic phosphobacteria

were enriched in sediments at all sampling sites (ex-

cept inorganic phosphobacteria at Site B).

Fig. 4. Size-fractionated activity of alkaline phosphatase in overlying and interstitial water of Lake Taihu (2004-09).


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Fig. 5. The stability of dissolved alkaline phosphatase of surface, overlying and interstitial water at Sites B and D of Lake Taihu in reverse micelles

(W0:14; pH:8.5; T:37) (2004-09).

Table 4 The bacterial abundance and respiration efficiency in all sampling sites of Lake Taihu (2004-09)

Sampling sitesAerobic bacteria

(103CFU/mL; 103CFU /g)Organic phosphobacteria

(CFU /mL; CFU/g)Inorganic phosphobacteria

(CFU /mL; CFU /g)Respiration efficiency

(mLCO2/g.h)Surface water of Site A 31 90

Overlying water of Site A 1.7 215

Sediment of Site A 1.9 30000 1500 2.46

Surface water of Site B 122 400 280

Overlying water of Site B 5.4 9

Sediment of Site B 1800 5.85Surface water of Site C 3.1

Overlying water of Site C 2.7 230

Sediment of Site C 440 20500 1900 2.55

Surface water of Site D 2.8 385 410

Overlying water of Site D 144 1100 600

Sediment of Site D 1620 27000 2900 4.80

, Undetectable.

4 Discussion

Eutrophic symptoms, such as high productivity

(Sites B and D) and Microcystissp. bloom (Site B) inLake Taihu, were closely related with internal load-

ings.

4.1 Internal loading and anaerobic release of

phosphorus

The concentrations of different phosphorus forms indifferent depths of sediments at the undredged sites

were markedly higher than those at the dredged sites


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Fig. 6. The stability of dissolved alkaline phosphatase of Lake Longyang and Lake Lianhua in micelles. (a) Different W0conditions; (b) differenttemperature conditions; (c) different pH conditions.

(Fig. 2) in January 2004. The lower redox potential insediments of the undredged sites was observed, for

example, the value was 102 mV in Site C, whereas

79 mV in Site A. In September, the concentration ofSRP in the interstitial water at Site B (withMicrocystis

sp. bloomed) was considerably low (Table 2), and dif-


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ferent phosphorus forms in surface sediment exhibited

a lower value than that at the undredged Site D, which

was approximate to the level at dredged Sites A and C(Fig. 3). In addition, higher respiration efficiency and

lower aerobic bacteria abundance in sediments of Site

B indicated the anoxic status (Table 4). Hence, the

anaerobic release of internal phosphorus loading may

be an important reason for the occurrence of Micro-

cystissp. bloom

4.2 High activity and stability of phosphatase

The APA in water column may act as an indicator

for the phosphorus status and eutrophication degree.The APA exhibited higher level in eutrophic lakes[34]and gulfs[35]. Contrarily, the APA and phosphorusconcentration were low in lake water with the growthof macrophytes[36,37]. Different size-fractionated APAin Site B generally showed the highest level, espe-cially the DAP in interstitial water (Fig. 4). Markedlyhigher activity and stability of DAP in micelles andSRP concentration in the interstitial water of Site D

were observed (Table 2, Fig. 5). Therefore, there was aclose relationship between phosphatase activity andeutrophication, which may be supported by the datafrom other lakes. In Lake Longyang, the SRP and

chlorophyll a concentrations were 0.418 mgL1 and

24.41gL1respectively, while in Lake Lianhua, the

responding values were 0.038 mgL1 and 10.23

gL1. Thus Lake Longyang was more eutrophic rela-

tive to Lake Lianhua. In addition, its size-fractionatedAPA was also higher. In Lake Longyang, APA associ-

ated with coarser (>3.0 m) and finer (0.453.0 m)

particles and DAP activities were 19.8, 6.2 and 35.5

nmolL1min1 respectively, while in Lake Lianhua

the responding values were 12.1, 0.3 and 24.2

nmolL1min1. It means that the eutrophic lake

showed not only significantly higher APA with differ-ent forms, but also significantly higher and more sta-

ble DAP activity in micelles (Fig. 6).W0 may regulate the micro-environment in water

pool of micelles and influence the enzymatic activity

and stability[38]. The thermal stability of enzyme de-

pends on the water content, decreasing when W0value

increases[39], which was consistent with the results inLake Longyang (Fig. 6(a)). In lower water content, the

reversed micelles are capable of rearranging to ac-

commodate a protein with dimensions higher than the

water pool due to the possibility of rearrangement of

the surfactant chains. For example, at W02.7 the mi-celles have a radius of inner cavity of 8, while the

cutinase can be represented by a sphere with 21.6.

This implies that upon encapsulation, the micelles are

enlarged to host the protein. Cutinase in such small

micelles is structurally immobilized which leads to a

stabilization through a rigidification of its structure,

since its secondary and teriary structures are preserved

from denaturation usually associated to an unfolding

process[40]. Hence, low W0coupled with high stability

is related with enzyme molecule. However, at low W0the stability of DAP in Lake Lianhua decreased, sug-gesting the difference of size of enzymatic molecule in

two lakes.

In three designed temperature conditions, DAP of

two lakes in micelles lost its activity completely in a

short time at 50(Fig. 6(b)). The polyphenol oxidase

activity decreased to the lowest level at 70. The

possible explanation is that high temperature can

modify the physical characteristics of micelles, and

those changes probably include the rearrangement ofsurfactant molecules influencing enzymatic conforma-

tion and activity[41,42]. In detail, solvation is necessary

for protein function. However, excessive solvation

may lead to the loss of the native protein structure.

This is more patent at high temperatures in which

many of the weak bonds that maintain the nativestructure are destabilized and solvated[30]. In the same

temperature condition, the stability of DAP of two

lakes in micelles represented evidently distinct feature

(Fig. 6(b)), implying the difference of enzymatic con-

formation.

In general, the stability of phosphatase in micelles

cannot be affected by pH[43], which was also proved in

our results (Fig. 6(c)). In the micellar microenviron-

ment, the ideas that pH of the initial buffer solution

changes upon micelle formation is widely spread, and

the ionogenic groups of the enzyme will be affected by

the microenvironment and their ionization state se-

verely influences the interaction with substrate and

inhibition by products[44]. For example, pH may acti-

vate Phosphatidylinositol-specific phospholipase bydisrupting ionogenic groups leading to a conforma-tional change[45]. Furthermore, some changes are veri-


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fied to pH, but they depend on the amino residues of

each protein[46]. Hence, the variation of enzymatic

activity with pH in micelles was related to ionogenicgroups. In the same pH conditions, there was distinctly

different activity and stability of DAP in two lakes

(Fig. 6(c)), indicating the different amino residues of

enzyme.

Therefore, in the lakes and some hypolimnion with

higher trophic levels, DAP showed higher activity and

stability. The variations in DAP stability in micelle, as

affected by different W0, temperature and pH condi-

tions, reflected the characteristics of molecular size,

conformation and active residues of the enzyme, sug-gesting the existence of isozymes in different lakes.There existed phosphatase isozymes dependent on pH

in the streams[47]. Extracellular enzymes with different

origins in seawater may hydrolyze structurally distinct

polysaccharides by different velocities, implying that

microbial communities in different habitats can pro-

duce extracellular enzymes with specific substrates

and similar functions [48]. This paper provided further

evidence for the occurence of dissolved phosphatase

isozymes from the view-point of micellar enzymology.In short, with higher activity and stability, DAP

isozymes could accelerate the eutrophication by

stimulating the release of internal phosphorus loading.

4.3 Microbial activity

Hypoxia may cause aerobic bacteria autolyze,

thereby resulting in cellular phosphorus release[49],which may occurred in the sediment of Site B. Thus,

the release of internal phosphorus was closely related

with anoxic status and the variation of microbial

community. In addition, among all the sampling sites,Site B with Microcystis sp. bloom showed the most

abundant organic and inorganic phosphobacteria in the

surface water and the lowest amounts of bacteria in

sediments (Table 4), suggesting that bacteria in the

surface water may migrate from the sediment. A pos-

sible explanation is that organic carbon produced by

bloom algae induces the mobility of bacteria, which is

positively related with particle organic matter[50]. It is

supposed that there exists a metabolic coupling be-

tween bacteria and bloom algae. Bacteria provide in-organic phosphorus through decomposition for algae

bloom, and in turn, the algae provide substantial or-

ganic carbon as bacterial carbon sources, reflecting a

mutuality between phytoplankton and bacteria, even

they compete with each other for inorganic phospho-rus[51]. Hence, the mobility of bacteria coupled with

the phosphorus release should be an additional content

of internal loadings.

5 Conclusions

In Lake Taihu, the undredged zone had the symp-

toms of eutrophication with high phosphorus loading

and the releasing potential under anaerobic conditions

in sediments. The phosphatase activity was closelyrelated with the degree of eutrophication in the lake.

The behavior of DAP in reverse micelle may reflect its

characteristics of molecular size, conformation and

active residues. High and stable activity of DAP, com-

bined with the varying population of aerobic, inor-

ganic and organic phosphorus bacteria, substantially

contributed to the internal phosphorus loading andpromoted its flux. Biological mechanism may act in

concert with physical and chemical factors to facilitate

the internal phosphorus release and the excess growth

of algae, which may accelerate the process of lake eu-

trophication.

Acknowledgements The authors would like to thank DrLi Lin, Wu Zhongxing and Peng Liang for their help insampling. This work was supported by the Chinese Acad-emy of Sciences (CAS) (Grant No. KZCX1-SW-12-II-02-02), and the National Natural Basic Research Program ofChina (Grant No.2002CB412304), the National NaturalScience Foundation of China (Grant No. 20177033). We arealso obliged for the funds (Grant No.2002AA601013).

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