hydrolytic and transport systems should form a closely related unit a ‘complex’ to ensure the efficient utilisation
of substrates. Although the transport of small molecules through
membranes is believed to be not a limiting step for the system Chrost, 1989; Mu¨nster and Chrost, 1990, both
components of a complex can become saturated. Compar- ison of the kinetics of the extracellular enzymeuptake
complex could give an insight into the ecology and substrate utilisation in aquatic and soil systems.
The highest extracellular enzymatic activities are usually found during the period after phytoplankton bloom when
algae are dying and being lysed e.g. Chrost, 1989. During this time bacterial uptake of the products of hydrolysis,
rather than algal exudates as during the bloom are believed to support metabolism and growth. Extracellular enzymes
may not have great importance during the period of active phytoplankton growth as most of the newly produced
organic carbon available to bacteria originates directly from the release of photosynthesis products Chrost and
Overbeck, 1990; Middelboe et al., 1995 and zooplankton grazing of algae Vrba et al., 1992. However, during the
period after the phytoplankton bloom, production of extra- cellular enzymes is the response of bacterioplankton to
carbon sources of different type which become available.
The objectives of the present study were to follow both the uptake of two readily utilisable substrates, glucose and
leucine, and the extracellular enzymatic activities of the b- glucosidase and leucine aminopeptidase, both during and
after the cyanobacterial bloom. Several other bacterioplank- ton parameters, such as biomass and production, phyto-
plankton biomass, chlorophyll concentration, and the physico-chemical properties of water were also followed.
2. Material and methods
Samples were collected over a period of a week from Lake Vo˜rtsja¨rv, a shallow, highly eutrophic lake in Central
Estonia area, 270 km
2
; maximum depth, 6 m; and mean depth, 2.8 m. Lake water was collected at the surface and
then at 1, 2, 3, 4, 5, 6 m depth 2 l of each sample using a Ruttner sampler. These samples were mixed in a sterile
plastic barrel in order to get vertically integrated plankton samples. The total number of bacteria was determined by
fluorescence microscopy on DAPI stained 0.22-mm black membrane Osmonics Inc., Livermore, USA filters Porter
and Feig, 1980. The productivity of bacteria was estimated using the
3
H-leucine incorporation method Simon and Azam, 1989. The colony forming units CFU were
measured as plate counts on standard method agar SMA, Becton Dickinson. Samples of 0.2–0.5 ml undiluted water
were inoculated on two replicate Petri dishes containing growth medium and incubated for 7 d at 20
8C Ott et al., 1997.
2.1. Kinetics of hydrolytic enzymes Extracellular enzyme activity was measured from the
increase in fluorescence due to the products of the non- fluorescent substrates. Methyl-umbelliferyl glucose MUF-
glucose and leucine amino-methylcoumarin Leu-AMC hydrolyses b-Gluc and LAP activities were measured
according to Hoppe 1983 and Chrost et al. 1986.
Fluorescence was measured at 450 MUF and 440 nm AMC for emission and at 365 MUF and 315 nm AMC
for excitation Perkin–Elmer 203 spectrofluorometer. Quantification of MUF and AMC was achieved by calibra-
tion with standard solutions MUF 30 nM–20 mM; AMC 50 nM–2 mM. Stock solutions of the substrates 2.5 and
25 mM MUF-glucose; 1 and 10 mM AMC were stored at 2258C. Triplicate water samples, 5 ml, were supplemented
with different amounts of the stock solution to give a 5 mM– 4 mM concentration range of MUF-glucose and 0.8–
200 mM of Leu-AMC. Samples were incubated for 4 h at 25
8C. Michaelis–Menten kinetics parameters of enzyme reactions K
M
and V
max
were calculated using non-linear regression analyses from original experimental data.
Kinetics parameters were used to estimate changes in enzyme activity.
2.2. Monomer substrate uptake kinetics Substrate uptake was measured in 5-ml duplicate water
samples by addition of different amounts of radiolabelled glucose 1–50 nM, 180 mCimmol, Sigma and leucine 3–
277 nM, 59.0 Cimmol, Amersham Ltd.: 1–50 and 3– 277 nM, respectively. Samples were incubated at 25
8C for 1 h leucine or 2 h glucose. Incubation was terminated by
adding formaline final conc. 2 vv and the samples were filtered through 0.2-mm pore-size cellulose acetate filters
Millipore. The vials were rinsed once with 1 ml and filtered six times with 1 ml of 0.8 NaCl. Kinetics para-
meters were calculated as for the enzyme reactions.
2.3. Other parameters Primary production PP
part
of phytoplankton and the release of phytoplankton products into the extracellular
dissolved fraction PP
diss
were estimated on the basis of the
14
C assimilation method Kisand et al., 1998. In brief, primary production PP was estimated in situ by NaH
14
CO
3
final activity 0.07 mCi ml
21
, VKI, Denmark assimilation. Samples were incubated for 2 h at six depths in the lake. For
measurements of phytoplankton PP PP
part
, water was filtered through membranes of 0.45 mm pore-size Millipore
HA, for measurements of dissolved PP PP
diss
the filtrate and total PP PP
tot
water was acidified pH ,2 by HCl
Niemi et al., 1983; Hilmer and Bate 1992; Lignell 1992. The radioactivity of water, filtrate and filters was assessed
using a scintillation counter LSC RackBeta 1211, Wallac, Finland. Non-photosynthetic carbon fixation was measured
in dark incubations and subtracted from light assimilation.
V. Kisand, H. Tammert Soil Biology Biochemistry 32 2000 1965–1972 1966
Samples for phytoplankton biomass were preserved and fixed with Lugol’s iodine. Species were identified and the
biomass was determined using an inverted plankton micro- scope Olympus IMT-2, magnification of 400 × Uter-
mo¨hl, 1958.
Algal pigments
chlorophyll a,
pheopigments were extracted with 90 vv acetone and analysed spectrophotometrically Strickland and Parsons,
1972. Zooplankton samples were preserved in the 4 vv formaldehyde solution and studied by conventional quanti-
tative analysis Kiselev, 1956. Chemical analyses were performed in depth integrated water samples, using the
methods described by Grasshoff et al. 1983.
2.4. Statistics For comparisons of independent groups, Wilcoxon signed
rank non-parametric tests were used. Comparing differences between single samples in one time series, known distribu-
tion Poisson distribution for counting measurements, normal distribution for others and independence of single
samples was assumed. Absolute difference between samples was compared using confidence intervals of each single
measurement.
3. Results
