Microbial Enzymatic Activities and Carbon Flux in the Adriatic Sea
487
fluorescence was measured with a Hitachi spectrofluorometer at 380 and 440 nm excitation and
emission wavelength, respectively. Velocity of
hydrolysis (v, ]lg C 1- 1 h- 1 ) for each concentration
of substrate was calculated, in terms of C content,
according to Hoppe (1993). The Vmax (maximum
velocity of hydrolysis) and Km (Michaelis constant) were obtained by plotting 11[8) versus IIv
using the Michaelis-Menten equation rearranged
in a linear form: Lineweaver-Burk transformation
(Michal 1983).
The respiration rates and the consequent
metabolic production of CO 2 were detected measuring the electron transport system activity
(ETS). The method is based on the conversion of
tetrazolium salt in formazan (Packard 1971) and
details of the procedure are quoted by La FerIa et
al. (1996). Results are reported as Vmax and converted into C by using a respiratory quotient
(R.Q.) of 1 (Vosjan and Nieuwland 1987). This
choice was done to better compare respiratory
rates with peptidase activity data.
Bacterial carbon biomass (BB) was calculated using a factor of 20 fgC/cell (Ducklow and
Carlson 1992) on estimates of bacterial abundance determined by DAPI staining method
(Porter and Feig 1980). In area B, bacterioplankton cell volumes and morphotypes were determined by the microphotographic technique,
using AO staining, according to La FerIa et al.
(1998).
Heterotrophic viable bacteria counts (MA)
were determined on Marine Agar 2216 (DIFCO),
by spread plate and incubation at 22°C for 7 days
(Zaccone et aL 1998).
Respiration, aminopeptidase and bacterial
biomass data have been statistically analysed by
ANOVA to test the data means for differences
and by Pearson correlation coefficient to measure the associations between pairs of variables.
In this paper, the concept of "potential activity" is taken into account because the rates are
expressed as Vmax.
Results
Ranges and mean values with standard deviation
of the aminopeptidase activity (AMP). respiratory carbon production (R). bacterial biomass (BB)
and viable heterotrophic bacteria (MA) together
with salinity and temperature, determined in the
two areas, are reported in Table 1. In the area A,
AMP increased in summer (1.5 times with
respect to winter), reaching maximum in June
96 (mean 9.97±1O.72]lg C 1-1 h- 1 ) and decreased
in winter (minimum in February 98, mean
4.86±8.60 ]lg C 1-1 h- 1 ). R also showed seasonal
changes since, in June 96, it was 4.5 fold higher
than February 97 and in June 97,6 fold higher
than February 98. Maximum occurred in June 97
(mean 2.29±3.20 ]lg C 1-1 h- 1 ) and minimum in
February 97 (mean 0.19±O.11 ]lg C 1-1 h- 1 ).
Bacterial biomass (BB) derived from DAPI direct
counts showed increasing values during the
whole research, from June 96 to February 98 (BB
mean range 9.22-43.90 llg C 1-1). The MA ranged
from 1.3 to 0.4x10 3 CFU/ml in February 97 and
98, respectively.
In area B, all the studied parameters showed
lower values with respect to area A, without any
relationships with seasons. Interannual differences were observed, with decreasing averaged
values of AMP from June 96 to June 97 (2.4 fold)
and from February 97 to February 98 (1.6 fold)
(Table 1). Increases in R were observed from June
96 to June 97 (3.8 times) and from February 97 to
February 98 (1.8 times); also BB increased (1.2
times both in summer and winter). The MA
showed higher values in winter and lower in
summer (6 and 2.3 fold, respectively).
The statistical analysis with ANOVA did not
reveal any significant differences for the studied
parameters between areas, seasons and years.
Mean biovolume changed between warm
and cold periods as reported in Table 2, with
larger cell volumes in summer (0.155 and 0.138
]lm 3 in June 96 and 97. respectively) and smaller
in winter (0.048 and 0.021 ]lm 3 , in February 97
and 98, respectively). Significant differences were
registered by ANOVA test comparing summer to
winter, and winter months to each other but no
difference resulted between the summer months.
Bacterial morphotypes also changed in different
seasons with the predominance of bacilli and
vibrios, together with spirilli, in summer while,
during winter, cocci prevailed.
Since most microbial activity was confined
to the upper 10 m (Zaccone et al. 1999), Fig. 2
reports the integrated values of AMP and R calculated in this depth range. In area A, AMP values generally showed a negative gradient from
near-shore to offshore; this decrease was not
observed for R. Overall, the AMP potentially
hydrolysed 108.9 mg C m- 2 h- 1 in warm months
and 54.6 mg C m- 2 h- 1 in cold months. The
487
fluorescence was measured with a Hitachi spectrofluorometer at 380 and 440 nm excitation and
emission wavelength, respectively. Velocity of
hydrolysis (v, ]lg C 1- 1 h- 1 ) for each concentration
of substrate was calculated, in terms of C content,
according to Hoppe (1993). The Vmax (maximum
velocity of hydrolysis) and Km (Michaelis constant) were obtained by plotting 11[8) versus IIv
using the Michaelis-Menten equation rearranged
in a linear form: Lineweaver-Burk transformation
(Michal 1983).
The respiration rates and the consequent
metabolic production of CO 2 were detected measuring the electron transport system activity
(ETS). The method is based on the conversion of
tetrazolium salt in formazan (Packard 1971) and
details of the procedure are quoted by La FerIa et
al. (1996). Results are reported as Vmax and converted into C by using a respiratory quotient
(R.Q.) of 1 (Vosjan and Nieuwland 1987). This
choice was done to better compare respiratory
rates with peptidase activity data.
Bacterial carbon biomass (BB) was calculated using a factor of 20 fgC/cell (Ducklow and
Carlson 1992) on estimates of bacterial abundance determined by DAPI staining method
(Porter and Feig 1980). In area B, bacterioplankton cell volumes and morphotypes were determined by the microphotographic technique,
using AO staining, according to La FerIa et al.
(1998).
Heterotrophic viable bacteria counts (MA)
were determined on Marine Agar 2216 (DIFCO),
by spread plate and incubation at 22°C for 7 days
(Zaccone et aL 1998).
Respiration, aminopeptidase and bacterial
biomass data have been statistically analysed by
ANOVA to test the data means for differences
and by Pearson correlation coefficient to measure the associations between pairs of variables.
In this paper, the concept of "potential activity" is taken into account because the rates are
expressed as Vmax.
Results
Ranges and mean values with standard deviation
of the aminopeptidase activity (AMP). respiratory carbon production (R). bacterial biomass (BB)
and viable heterotrophic bacteria (MA) together
with salinity and temperature, determined in the
two areas, are reported in Table 1. In the area A,
AMP increased in summer (1.5 times with
respect to winter), reaching maximum in June
96 (mean 9.97±1O.72]lg C 1-1 h- 1 ) and decreased
in winter (minimum in February 98, mean
4.86±8.60 ]lg C 1-1 h- 1 ). R also showed seasonal
changes since, in June 96, it was 4.5 fold higher
than February 97 and in June 97,6 fold higher
than February 98. Maximum occurred in June 97
(mean 2.29±3.20 ]lg C 1-1 h- 1 ) and minimum in
February 97 (mean 0.19±O.11 ]lg C 1-1 h- 1 ).
Bacterial biomass (BB) derived from DAPI direct
counts showed increasing values during the
whole research, from June 96 to February 98 (BB
mean range 9.22-43.90 llg C 1-1). The MA ranged
from 1.3 to 0.4x10 3 CFU/ml in February 97 and
98, respectively.
In area B, all the studied parameters showed
lower values with respect to area A, without any
relationships with seasons. Interannual differences were observed, with decreasing averaged
values of AMP from June 96 to June 97 (2.4 fold)
and from February 97 to February 98 (1.6 fold)
(Table 1). Increases in R were observed from June
96 to June 97 (3.8 times) and from February 97 to
February 98 (1.8 times); also BB increased (1.2
times both in summer and winter). The MA
showed higher values in winter and lower in
summer (6 and 2.3 fold, respectively).
The statistical analysis with ANOVA did not
reveal any significant differences for the studied
parameters between areas, seasons and years.
Mean biovolume changed between warm
and cold periods as reported in Table 2, with
larger cell volumes in summer (0.155 and 0.138
]lm 3 in June 96 and 97. respectively) and smaller
in winter (0.048 and 0.021 ]lm 3 , in February 97
and 98, respectively). Significant differences were
registered by ANOVA test comparing summer to
winter, and winter months to each other but no
difference resulted between the summer months.
Bacterial morphotypes also changed in different
seasons with the predominance of bacilli and
vibrios, together with spirilli, in summer while,
during winter, cocci prevailed.
Since most microbial activity was confined
to the upper 10 m (Zaccone et al. 1999), Fig. 2
reports the integrated values of AMP and R calculated in this depth range. In area A, AMP values generally showed a negative gradient from
near-shore to offshore; this decrease was not
observed for R. Overall, the AMP potentially
hydrolysed 108.9 mg C m- 2 h- 1 in warm months
and 54.6 mg C m- 2 h- 1 in cold months. The