116
M. Mingazzini
Materials and Methods
Algal Cultures
Different algal species were used to characterize
the organic matter produced and released in the
extracellular medium. Four diatom species:
Skeletonema costatum, Nitzschia (renamed
Cylindrotheca) closterium, Chaetoceros Spa and
Navicula sp., isolated from North Adriatic Sea,
were cultured in the laboratory. While S. costatum and N. closterium were collected in summer
1991 from mucilage samples (Mingazzini et a1.
1995), Navicula and Chaetoceros were isolated
from water samples respectively collected in
September 1994 and May 1995 at nearshore
Cesenatico. The monospecific cultures were
grown in MAAP medium (EPA 1974) prepared
using oligotrophic seawater enriched with inorganic nutrients from the medium stock solutions (Mingazzini et al. 1995). Cultures were
kept in controlled conditions at 20±1°C temperature, at 3000-lux light intensity. with a
light:dark cycle of 14:10 hours. Natural phytoplankton from the marine sample stations (see
below) was also used to monitor the extracellular production. The unmtered seawater samples
(100 ml) containing the natural community
were incubated without any enrichment and
kept in the same conditions used for the monospecific cultures.
Differently aged cultures of S. costatum (0 to
40 days from inoculum) were used to monitor
the trend of production and accumulation of the
fluorescing compounds released in the different
growth phases. The same experimental framework was applied to the natural phytoplankton
cultures, starting from the incubation time 0 of
the samples.
For the fluorimetric characterization of the
extracellular medium, 5 to 10 ml-subsamples
were taken from the diatom as well as from the
natural phytoplankton cultures. All samples (for
EOM as well as for natural DOM determinations)
were mtered on Millipore HA OA5 Jll11 fUters
under reduced pressure ( <50 mm Hg) in order to
separate the EOM from the biomass, avoiding
breakage of cells. Each filter was rinsed in two
ways before use. First. prefiltered Ultrapure
water, and secondly. a part of the same sample
were passed through each fUter before collecting
the fUtrate.
Sampling Stations
The Po River delta, as the main contributor of
terrestrial DOM. providing over 50% of the
freshwater input to the North Adriatic basin, was
investigated. The map in Fig. 1 shows the study
area and the location of sampling stations. One
riverine water sample (Po) was collected dose to
the river mouth, in Po della Pila, which is the
main channel of the Po River delta. Seven marine
water samples (1 to 7) were taken from an
onshore-offshore transect, starting just outside
from the river mouth, at salinities ranging from 8
to 32.2 PSu.
The samples were collected from surface
waters (0.5 m) by the DAPHNE II oceanographic
vessel of the Emilia-Romagna Region. The sampling was performed within one day (21 June
1995) and the water samples (for DOM determinations) were mtered (see above) and transferred to the laboratory in dark-refrigerated conditions. All the DOM sample analyses were performed within 36 h after sampling.
This sampling represents a part of the surveys carried out within the PRISMA 1 Project
(Research Programme for the Adriatic Sea). A
number of chemical-physical determinations
was made within the project, and DOM was also
investigated by DOC analyses performed on the
same samples (Pettine et al. 1998).
Spectrofluorimetric Analyses
The synchronous fluorescence technique (VoDinh 1978), which involves the simultaneous
scanning of the excitation and emission monochromators keeping a constant wavelength offset
(.1\.\), was adopted in order to resolve the mixture
of chromatophoric components of the DOM.
This technique has been explored in the last
years in different fields of application (Cabaniss
and Shuman 1987; Vodacek 1989; Senesi 1990; De
Souza Sierra et al. 1994; Ahmad and Reynolds
1995; Galapate et al.1998). The choice of both the
range of excitation wavelengths and the interval
.1\.\ should be determined for each application. In
multicomponent mixtures of natural DOM samples the capacity to discriminate between fluorophores having different origin but similar fluorescence properties makes particularly difficult
the choice of LU. (De Souza Sierra et a1. 1994). In
the present study the instrumental parameters
M. Mingazzini
Materials and Methods
Algal Cultures
Different algal species were used to characterize
the organic matter produced and released in the
extracellular medium. Four diatom species:
Skeletonema costatum, Nitzschia (renamed
Cylindrotheca) closterium, Chaetoceros Spa and
Navicula sp., isolated from North Adriatic Sea,
were cultured in the laboratory. While S. costatum and N. closterium were collected in summer
1991 from mucilage samples (Mingazzini et a1.
1995), Navicula and Chaetoceros were isolated
from water samples respectively collected in
September 1994 and May 1995 at nearshore
Cesenatico. The monospecific cultures were
grown in MAAP medium (EPA 1974) prepared
using oligotrophic seawater enriched with inorganic nutrients from the medium stock solutions (Mingazzini et al. 1995). Cultures were
kept in controlled conditions at 20±1°C temperature, at 3000-lux light intensity. with a
light:dark cycle of 14:10 hours. Natural phytoplankton from the marine sample stations (see
below) was also used to monitor the extracellular production. The unmtered seawater samples
(100 ml) containing the natural community
were incubated without any enrichment and
kept in the same conditions used for the monospecific cultures.
Differently aged cultures of S. costatum (0 to
40 days from inoculum) were used to monitor
the trend of production and accumulation of the
fluorescing compounds released in the different
growth phases. The same experimental framework was applied to the natural phytoplankton
cultures, starting from the incubation time 0 of
the samples.
For the fluorimetric characterization of the
extracellular medium, 5 to 10 ml-subsamples
were taken from the diatom as well as from the
natural phytoplankton cultures. All samples (for
EOM as well as for natural DOM determinations)
were mtered on Millipore HA OA5 Jll11 fUters
under reduced pressure ( <50 mm Hg) in order to
separate the EOM from the biomass, avoiding
breakage of cells. Each filter was rinsed in two
ways before use. First. prefiltered Ultrapure
water, and secondly. a part of the same sample
were passed through each fUter before collecting
the fUtrate.
Sampling Stations
The Po River delta, as the main contributor of
terrestrial DOM. providing over 50% of the
freshwater input to the North Adriatic basin, was
investigated. The map in Fig. 1 shows the study
area and the location of sampling stations. One
riverine water sample (Po) was collected dose to
the river mouth, in Po della Pila, which is the
main channel of the Po River delta. Seven marine
water samples (1 to 7) were taken from an
onshore-offshore transect, starting just outside
from the river mouth, at salinities ranging from 8
to 32.2 PSu.
The samples were collected from surface
waters (0.5 m) by the DAPHNE II oceanographic
vessel of the Emilia-Romagna Region. The sampling was performed within one day (21 June
1995) and the water samples (for DOM determinations) were mtered (see above) and transferred to the laboratory in dark-refrigerated conditions. All the DOM sample analyses were performed within 36 h after sampling.
This sampling represents a part of the surveys carried out within the PRISMA 1 Project
(Research Programme for the Adriatic Sea). A
number of chemical-physical determinations
was made within the project, and DOM was also
investigated by DOC analyses performed on the
same samples (Pettine et al. 1998).
Spectrofluorimetric Analyses
The synchronous fluorescence technique (VoDinh 1978), which involves the simultaneous
scanning of the excitation and emission monochromators keeping a constant wavelength offset
(.1\.\), was adopted in order to resolve the mixture
of chromatophoric components of the DOM.
This technique has been explored in the last
years in different fields of application (Cabaniss
and Shuman 1987; Vodacek 1989; Senesi 1990; De
Souza Sierra et al. 1994; Ahmad and Reynolds
1995; Galapate et al.1998). The choice of both the
range of excitation wavelengths and the interval
.1\.\ should be determined for each application. In
multicomponent mixtures of natural DOM samples the capacity to discriminate between fluorophores having different origin but similar fluorescence properties makes particularly difficult
the choice of LU. (De Souza Sierra et a1. 1994). In
the present study the instrumental parameters