3. Results
3.1. Field investigations Alkaline phosphatase and peptidase activities in the
euphotic zone of the Gotland Basin in July 1993 are shown in Fig. 1. An inverse relationship between the extra-
cellular enzymes and their respective nutrients was observed. In the upper 12 m, phosphate concentrations
were at the detection limit 0–0.02 mM and increased to 0.35 mM at 21 m. Alkaline phosphatase activity reached a
maximum of 300 nM h
21
at 7 m and decreased to 50 nM h
21
in 21 m. The correlation coefficient between phosphate and alkaline phosphatase activity was r
20 :
85
n 9; p , 0
: 01
: Peptidase activity varied between
65 and 73 nM h
21
in the upper 12 m and decreased drasti- cally to 17 nM h
21
at greater depths. Dissolved inorganic nitrogen DIN concentrations increased in the same depth
profile, from 0.1–0.2 mM in the upper 12 m to 0.45 mM at 21 m. DIN and peptidase activity correlated with a coeffi-
cient of r 20
: 78
n 9; p
, 0 :
05 :
In the Pomeranian Bight, DIN decreased with increasing distance from the fresh water input Bodungen et al., 1995;
Nausch et al., 1999. Within these gradients, peptidase activities showed an inverse relationship to DIN and a
DIN threshold concentration of 3–4 mM could be detected for this relationship. At higher DIN concentrations, the
peptidase activity remained low and independent from the nutrient concentrations. The correlation coefficient for
the data shown in Fig. 2 was r 0
: 43
n 62; p
, 0 :
01 :
The correlation coefficient increased to r 0 :
55
n 22; p
, 0 :
01 if only DIN concentrations below 4 mM were applied.
With increasing alkaline phosphatase activities, up to a V
max
of 200 nM h
21
, the peptidase activity also increased. At higher alkaline
phosphatase activities, the peptidase
M. Nausch, G. Nausch Soil Biology Biochemistry 32 2000 1973–1983 1976
Fig. 2. Peptidase activities in relation to dissolved inorganic nitrogen concentrations in the Pomeranian Bight.
Fig. 3. Relationship between the specific peptidase and alkaline phosphatase activities in surface waters of the Pomeranian Bight. A third level polynominal function
y 10
27
x
3
2 10
24
x
2
1 0 :
0483x 2 2
: 0529 gave the best description of the relationship between both activities.
activities remained constant. The correlation coefficient between the two activities was r 0
: 68
n 18; p
: 01
: Using a curve fitting programme of Excel-software,
the best description of this relationship was obtained with a third level polynominal function Fig. 3. The same relation-
ship between alkaline phosphatase and peptidase activity was also found in the Gotland Basin, where the peptidase
activity did not increase any further when phosphatase activities increased higher than 250 mM h
21
. In this region, the correlation coefficient between both parameters was r
: 82
n 9; p 0
: 01
: 3.2. Mesocosm experiments
In the mesocosm experiments, a nutrient gradient was established by phytoplankton and bacterial growth. The
effects of phosphate and nitrate on alkaline phosphatase and peptidase activity were investigated by the addition of
phosphate and nitrate when alkaline phosphatase and pepti- dase activity was high and inorganic nutrients were low. In
Figs. 4 and 5, the course of phosphate, DIN and specific enzyme activities are shown before and after nutrient addi-
tion. Enzyme activity per volume, e.g. V
max
nM h
21
had the same pattern, but the effect of nutrient supply is more
obvious with the specific activities because nutrient treat- ment was followed by phytoplankton and bacterial growth.
The increasing quantity of enzyme-producing organisms was accompanied by an increase in enzyme activity.
Using the equation T
t S
t × B
t T, total activity per litre water; S, specific enzyme activity; B, phytoplankton or
bacterial biomass; t, time, the extent to which the change of V
max
can be attributed to the increase of biomass was
M. Nausch, G. Nausch Soil Biology Biochemistry 32 2000 1973–1983 1977
Fig. 4. Phosphate concentrations lines and specific alkaline phosphatase activity bars in mesocosm experiments.
Fig. 5. Dissolved inorganic nitrogen concentrations lines and specific peptidase activity bars in mesocosm experiments.
calculated. Only about 2 of the increase of V
max
of alkaline phosphatase activity can be explained by the increase of
phytoplankton and bacteria in the control, 38 in the meso- cosm after nitrate supply and 83 after nitrate plus phos-
phate treatment. The increase of V
max
of peptidase activity can be attributed to bacterial growth in the mesocosm with-
out nutrient addition and in the mesocosm with nitrate addi- tion by about 28. In the mesocosm with nitrate plus
phosphate additions, bacterial growth accounted for only about 15 of the development of V
max
of peptidase activity. The experiments can be divided into two phases: those
before and those after nutrient addition. In the first phase, the increase of alkaline phosphatase and peptidase activity
was parallel to the decrease in inorganic nitrogen and phos- phate concentrations. The peptidase activity increased 5- to
7-fold whereas the alkaline phosphatase activity increased between 29- and 65-fold. The phytoplankton biomass Chl
a, total organic phosphorus and nitrogen increased parallel to the enzyme activities Tables 1–3. However, the closest
correlation coefficient
r 0 :
90; n 18; p , 0
: 01 was
between peptidase and alkaline phosphatase activity. There were also correlations between the peptidase activity
and total particulate and dissolved organic nitrogen
r
: 61
; n 18
; p
, 0 :
01 and to a lesser extent with total particulate and dissolved organic phosphorus
r 0
: 48
; n 18
; p
, 0 :
05 Fig. 6. In the second phase, the alkaline phosphatase activity
reacted as expected, with a decrease following the addition of phosphate. The addition of nitrate alone resulted in an
increase of V
max
Table 2, but the specific alkaline phospha- tase activity was reduced Fig. 4. The peptidase activity
was highest after simultaneous treatment with nitrate and phosphate and correlated better with Chl a and organic
phosphorus than with organic nitrogen concentrations
M. Nausch, G. Nausch Soil Biology Biochemistry 32 2000 1973–1983 1978
Table 1 Development of alkaline phosphatase activity APA and peptidase activity and different biomass parameters in mesocosm experiments without nutrient
addition Parameter
Incubation time days 3
6 8
13 15
17 20
NO
3
mM 5.74
3.87 2.06
1.91 0.16
0.05 0.1
0.28 PO
4
mM 1.02
0.75 0.5
0.5 0.07
0.07 0.07
0.04 APA nM h
21
0.0 0.5
1.6 1.5
1.9 3.6
3.0 4.4
Spec. APA nM mg
21
Chl a h
21
0.1 0.1
0.7 0.8
1.8 2.9
2.8 3.8
Peptidase activity nM h
21
5.0 6.7
4.4 12.9
32.0 44.0
33.3 63.3
Spec. peptidase activity fmol cell
21
h
21
2.6 2.3
n.d. 3.3
n.d. 22.0
15.3 20.9
Chlorophyll a mgl
21
1.8 4.7
2.0 2.0
1.1 1.2
1.8 1.3
Dissolved org. phosphorus mM 0.2
0.3 0.6
0.6 0.7
0.5 0.7
0.6 Particulate org. phosphorus
mM 0.2
0.5 0.4
0.3 0.3
0.5 0.3
0.2 Dissolved org. nitrogen mM
18.9 19.1
20.3 20.4
20.3 19.8
27.4 25.7
Particulate org.nitrogen mM 3.8
4.8 4.7
4.6 5.2
6.2 7.7
6.5 Bacterial counts cells ml
21
1.9 × 10
6
2.9 × 10
6
n.d. 4.2 × 10
6
n.d. 2.6 × 10
6
2.2 × 10
6
3.0 × 10
6
Table 2 Development of alkaline phosphatase activity APA and peptidase activity and different biomass parameters in mesocosm experiments after nitrate addition at
day 15 value immediately after nutrient supply Parameter
Incubation time days 3
6 8
13 15 addition
17 20
NO
3
mM 5.6
4.12 0.63
0.48 0.08
0.046.77 10.5
14.92 PO
4
mM 1.2
0.77 0.44
0.45 0.11
0.070.06 0.05
0.04 APA nM h
21
0.1 0.5
1.1 2.0
2.1 2.783.97
7.7 14.1
Spec. APA nM mg
21
Chl a h
21
0.1 0.1
0.3 0.6
2.1 2.4
1.4 2.2
Peptidase activity nM h
21
6.3 8.1
4.1 20.6
30.6 39.7
68.5 179.8
Spec. peptidase activity fmol cell
21
h
21
4.6 3.2
n.d. 4.8
n.d. 15.0
22.0 43.0
Chlorophyll a mgl
21
1.7 5.5
3.3 1.3
1.0 1.2
6.8 7.6
Dissolved org. phosphorus mM 0.2
0.4 0.6
0.7 0.8
0.5 0.7
0.6 Particulate org. phosphorus mM
0.7 0.4
0.4 0.4
0.4 0.6
0.4 0.3
Dissolved org. nitrogen mM 17.4
19.6 19.6
22.1 22.6
19.5 25.8
30.2 Particulate org.nitrogen mM
3.1 5.3
5.7 4.6
5.4 5.3
10.9 12.2
Bacterial counts cells ml
21
1.3 × 10
6
2.5 × 10
6
n.d. 4.2 × 10
6
n.d. 3.0 × 10
6
3.2 × 10
6
4.2 × 10
6
Table 3. Correlation coefficients between peptidase activ- ity and Chl a, as well as total organic phosphorus, were r
: 75
n 8; p 0
: 05
: The relationship between peptidase
activity and total organic nitrogen
r 0 :
32 was not signif- icant. Bacterial growth was similar in both mesocosms, plus
nitrate only and plus nitrate and phosphate. Phytoplankton biomasses and dissolved organic phosphorus levels were
higher after combined treatment with phosphate and nitrate than with nitrate treatment alone Tables 2 and 3.
3.3. Stimulation experiments In these experiments, it was investigated whether alkaline
phosphatase and phosphate supply could influence the bacterial peptidase activity. Treatment of 0.8 mm-filtered
seawater with alkaline phosphatase resulted in the stimula- tion of peptidase activity. The peptidase activity increased
with the incubation time, and reached a maximum at 72 or 96 h. At 96 h, activity was 6-fold higher than the control.
Activity was reduced at longer incubation times Fig. 7. The activity of the added alkaline phosphatase decreased
with increasing peptidase activity. The specific peptidase activity showed the same pattern. The bacterial counts
increased during the incubation time, reaching a maximum at 72 or 96 h and then declining at 120 h Table 4.
Stimulation of peptidase activity depended on the quantity of added alkaline phosphatase Fig. 8. The highest peptidase
activity was observed at 1 mg l
21
alkaline phosphatase- protein and a measured phosphatase activity of 15 mM h
21
. Higher levels 2 mg l
21
caused a slightly lower peptidase
M. Nausch, G. Nausch Soil Biology Biochemistry 32 2000 1973–1983 1979
Table 3 Development of alkaline phosphatase activity APA and peptidase activity and different biomass parameters in mesocosm experiments after phosphate plus
nitrate addition at day 15 value immediately after nutrient supply Parameter
Incubation time days 3
6 8
13 15 addition
17 20
NO
3
mM 5.68
4.12 2.21
2.02 0.08
0.055.95 9.4
8.93 PO
4
mM 0.97
0.67 0.47
0.41 0.01
0.010.8 1.93
3.75 APA nM h
21
0.0 0.4
1.0 1.1
2.6 3.2
4.3 5.9
Spec. APA nM mg
21
Chl a h
21
0.0 0.1
0.5 0.5
1.7 2.1
0.6 0.6
Peptidase activity nM h
21
4.8 8.0
3.5 16.9
35.7 33.2
140.3 521.7
Spec. peptidase activity fmol cell
21
h
21
2.4 3.1
n.d. 3.8
n.d. 17.0
46.3 136.0
Chlorophyll a mgl
21
2.0 5.0
2.0 2.0
1.6 1.5
8.0 9.9
Dissolved org. phosphorus mM 0.2
0.4 0.6
0.8 0.9
0.6 1.5
1.3 Particulate org. phosphorus mM
0.3 0.2
0.2 n.d.
0.3 0.3
0.6 1.5
Dissolved org. nitrogen mM 17.6
20.1 18.9
19.9 22.0
21.0 25.1
28.9 Particulate org.nitrogen mM
3.2 6.1
4.4 4.3
7.9 7
13.1 15.5
Bacterial counts cells ml
21
1.9 × 10
6
2.5 × 10
6
n.d. 4.4 × 10
6
n.d. 2.6 × 10
6
3.0 × 10
6
3.8 × 10
6
Fig. 6. Relationships between peptidase activity and total organic phosphorus, and nitrogen and alkaline phosphatase activity in the first part of the mesocosm experiments, during a period of declining inorganic nutrients, and before nutrient addition. The correlation coefficients are: peptidase activity-total organic
phosphorus r 0 :
48 ;
n 18 ;
p 0 :
05; peptidase activity-total organic nitrogen r 0 :
61 ;
n 18 ;
p 0 :
01; peptidase activity-alkaline phosphatase activity r 0
: 90
; n 18
; p 0
: 01
:
activity. The specific activity increased during the incubation time. At 72 h-incubation, the specific peptidase activity was
higher at 1 mg l
21
and 0.15 mg l
21
alkaline phosphatase than at 2 mg l
21
alkaline phosphatase. At 96 h, the differences between the concentrations were not so clear.
The supply of phosphate did not influence V
max
of pepti- dase activity up to 48 h. Later, a suppressive effect of phos-
phate on the V
max
of the peptidase was observed Table 5. The specific peptidase activity did not differ significantly
from the control and remained at a constant level during the incubation period. In addition, alkaline phosphatase
activity was not influenced by phosphate treatment. In these
experiments phosphate
concentrations ranged
between 0.45 and 0.21 mM. Bacterial development was the same in the control and after phosphate treatment.
4. Discussion
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