to maintain primary production Wasmund et al., 2000. We investigated both peptidase and alkaline phosphatase activ-
ity in nutrient gradients in the Baltic Sea, and addressed the question of regulatory factors. It is assumed that peptidases
are regulated by the availability of polymeric nitrogen substrates, such as proteins and large polypeptides, and
provide low molecular weight compounds for rapid assim- ilation by bacteria Hollibaugh and Azam, 1983; Billen,
1991; Chro´st 1991. However, the influence of inorganic nitrogen on the peptidase activity of pure bacterial cultures
is shown by Priest 1984 and was shown in the environment only by Chro´st 1991 in lakes. In contrast, the regulation of
alkaline phosphatase in aquatic environments by phosphate availability has been described extensively e.g. Halemejko
and Chro´st, 1984; Gage and Gorham, 1985; Paasche and Erga, 1988; Hernandez et al., 1993. We investigated the
influence of nitrate, phosphate and alkaline phosphatase on peptidase activity in an attempt to explain the observed
behaviour of peptidases in nutrient gradients in marine environments.
2. Material and methods
2.1. Field investigations Field investigations were carried out in the Gotland Basin
57 8 18.425
N and 020 8 04.52
E from June 27 to July 3, 1993. Drift experiments on the salinity gradient were
performed in the Pomeranian Bight in June 1994 and 1995. Inorganic nutrient gradients have different origins in
these areas. In the euphotic zone of the Gotland Basin, the nutrient gradient is caused exclusively by the uptake of
nutrients by phytoplankton. The salinity during the investi- gations ranged between 7.04 and 7.14 with depth, whereas
the temperature decreased from 14.2
8C at 1.5 m to 3.68C at 25 m. In contrast, in the Pomeranian Bight, nutrient-
enriched fresh water comes from the Odra River and is mixed with water of higher salinity and a lower nutrient
content from the open Baltic Sea Bodungen et al., 1995. Consumption of nutrients by phytoplankton in the growth
season reduces the nutrient concentration concomitantly. During the investigation, salinity increased from about 3,
near the river mouth, to 7.5 after the mixing of both water bodies. The temperature ranged between 15 and 19
8C. The Bight is relatively shallow, with a mean water depth of
13 m. Water was collected at depths of 1.5, 4.5, 7, 9, 12, 15,
16.5, 18 and 21 m in the euphotic zone of the Gotland Basin, and at 1.5 m from the surface and 1 m from the bottom in the
Pomeranian Bight. Water samples were taken with a rosette sampler Hydrobios Apparatebau GmbH, Kiel-Holtenau,
Gemany combined with a probe for measuring the conduc- tivity, temperature, density CTD Seabird SBE 911, Belle-
vue, WA, USA and chlorophyll fluorescence Haardt- Optik, Mikroelektonik, Kiel, Germany.
2.2. Mesocosm experiments Mesocosm experiments were performed in January and
February 1998, over a period of 20 days. Three 100-l meso- cosms, filled with nutrient-rich winter water from the
Arkona Basin Baltic Sea, were illuminated permanently with 500 W halogen lamps light intensity 200 mE s
21
m
22
to induce phytoplankton development and nutrient gradi- ents. After the depletion of nutrients and increased alkaline
phosphatase and peptidase activity at day 15, 7 mM potas- sium nitrate MERCK 5065 was added to the first meso-
cosm, and 7 mM potassium nitrate plus 1 mM potassium dihydrogen phosphate MERCK 4873 were added to the
second mesocosm. The same amounts of nutrients were added each day after the first supply to prevent nutrient
depletion by phytoplankton uptake. The third mesocosm control did not receive any nutrients. The incubation
temperature was 10
8C. Inorganic nutrient concentrations were determined daily. Extracellular enzyme activities,
chlorophyll a Chl a, bacterial numbers, organic phos- phorus and nitrogen were measured every two or three days.
2.3. Stimulation experiments Four successive stimulation experiments were performed
in January and February 1999 using water from the Arkona Basin, which was stored in a 100-l mesocosm. To induce
phytoplankton development and nutrient depletion, the mesocosm was stored at 5
8C and illuminated, as described above. However, the phytoplankton development was weak
and nutrients decreased slowly. Inorganic nutrient concen- trations were 0.45 mM PO
4
and 5.42 mM NO
3
1 NO
2
during the first experiment and 0.07 mM PO
4
and 4.65 mM NO
3
1 NO
2
during the last experiment. In these experi- ments, alkaline phosphatase SIGMA P7640 and potassium
dihydrogen phosphate PO
4
MERCK 4873 were added to 2 l of filtered 0.8 mm Arkona Basin water contained in
Duran Bottles. Final concentrations were 2, 1, or 0.15 mg l
21
for alkaline phosphatase and 1 mM of PO
4
. The bottles were incubated at 11
8C in the dark and aerated with air passed through a 0.2 mm filter. The peptidase activ-
ity and the activity of added alkaline phosphatase were esti- mated every day for 5 days.
2.4. Nutrient and chlorophyll analysis Phosphate, nitrite and nitrate were determined with stan-
dard colorimetric methods, according to Rohde and Nehring 1979 and Grasshoff et al. 1983. Dissolved phosphorus
and nitrogen, as well as total phosphorus and nitrogen were oxidised simultaneously with persulphate in alkaline
medium Grasshoff et al., 1983 followed by phosphate and nitrate determination using an autoanalyser system
Alliance Instruments GmbH, Friedrichsdorf, Germany. Dissolved organic nutrients were calculated as the differ-
ence between total dissolved phosphorus or nitrogen and inorganic
phosphate or
nitrogen, respectively.
The
M. Nausch, G. Nausch Soil Biology Biochemistry 32 2000 1973–1983 1974
particulate organic nutrient content is the difference between total phosphorus or nitrogen and the dissolved
fraction. Chl a was estimated fluorometrically excitation wave-
length 450, and emission 670 nm after filtration Whatman GFF-filter and extraction in 90 acetone UNESCO,
1994.
2.5. Bacterial enumeration Water samples for bacterial counting were preserved with
formaldehyde final concentration of 0.5 vv Kepner and Pratt, 1994. Sub-samples, 5–10 ml for field experiments
and 1–5 ml for stimulation experiments depending on the bacterial concentration were filtered onto 0.2 mm black
Nuclepore filters. The filters were stained with a 10 mg l
21
4,6-diaminidino-2-phenylindol DAPI solution for 5 min and mounted with fluorescent free immersion oil Cargille
oil. Cells were counted with an epifluorescence microscope Zeiss-Axioskop combined with an image analysis system
Photometrix GmbH, Mu¨nchen, Germany and the compu- ter software “IP Lab Spectrum” Signal Analytic Corp.,
Vienna, USA. Twenty fields or more were counted to deter- mine a minimum number of 400 cells. Cells per ml were
calculated according to Turley 1993 and cell volumes according to Sieracki et al. 1989.
2.6. Enzyme activities Peptidase
and alkaline
phosphatase activity
were measured according to Hoppe 1993, with leucine-4-
methyl-7-coumarylamide hydrochloride
Leu-MCA FLUKA 61888 and 4-methylumbelliferyl phosphate
MUF-P SIGMA M 8883 as substrates. The maxi- mum potential rate of hydrolysis V
max
was measured at concentrations of 250 mM MUF-P or Leu-MCA. The
incubation was performed using cuvettes in triplicates and in the dark. Incubation temperatures were in situ
in field investigations, or the temperatures at which the other
experiments were
performed. Fluorescence
measurements were made using a spectrofluorometer SFM 25, KONTRON Instruments GmbH, Neufahrn,
Germany at 364 nm excitation and 445 nm emission wave- lengths. The V
max
describes the enzyme activity in a certain volume of water in nmole l
21
h
21
nM h
21
, whereas the ‘specific’ peptidase enzyme activity is calculated per bacter-
ial cell and is given in amol cell
21
h
21
. Because phytoplank- ton is the main producer of alkaline phosphatase during its
growth phase, the enzyme activity in mesocosm experi- ments is related to the phytoplankton biomass as specific
alkaline phosphatase activity and is given in nM mg
21
Chl a h
21
. All calculations and statistical analyses were performed
with computer software Microsoft Excel 97.
M. Nausch, G. Nausch Soil Biology Biochemistry 32 2000 1973–1983 1975
Fig. 1. A Alkaline phosphatase activity and phosphate concentrations, and B peptidase activity and dissolved inorganic nitrogen in the euphotic zone of the Gotland Basin in July 1993. Standard deviations of alkaline phosphatase activity values are 4 for the depths 1.5–15 m and 14 for the other depths. Standard
deviations for the peptidase activity values were 6.5 1.5–15 m and 23 .15 m.
3. Results
