MATERIALS AND METHODS
Degradation of petroleum in continuous culture.
A crude oil (7.5 g.l -1 ) Asthart type is degraded continuously in fermentor containing
filtered sea water supplemented in PO4 (0.4 mM) at pH 8 and 30°C, with a dilution rate of
0.05 h - 1 . The fermentor is inoculated with a mixed bacterial culture which is composed of
8 strains isolated from sea water foams in hydrocarbons polluted areas. In such experimental conditions, the percentage of degraded crude oil is 83 %. The used medium is
centrifuged at 15 000 rpm for 15 mn. Remaining bacteria and most of residual petroleum
are removed from the used medium through 0.2 pm Millipore membrane before its
introduction in the culture medium of the test algae.
Test algae
Both algae are common species from the Gulf of Marseille : Phaeodactylum tricornutum
has been chosen because much is known about its physiology and its growth, even with
respect to hydrocarbon experiments (Lacaze and Villedon de Naide, 1976) ; (Lacaze,
1978 ; Kusk, 1981 a,b) : Prorocentrum micans is a Dinoflagellate often found in surface
slicks and possibly in connection with high lipid level in surface water (Kattner and
Brockmann, 1978 ; Cassie, 1981 ; De Souza Lima, 1981). Its growth is slower than that of
P. tricornutum.
Culture medium of algae
Cultures are grown in 40 ml test tubes closed with cotton plugs allowing volatiles to
escape. Tubes are successively filled up with :
- nutrients (Antia and Cheng algae medium)
- various percentages of the fermentor used medium (adjusted to 100 % in filtered sea
water). When required, lipids have been extracted from the used medium. This allows
lipids, lipid free used medium and untreated used medium to be tested separately on
phytoplankton. Each test is carried out with suitable control. The lipids are extracted
using diethyl ether after acidification of the medium (pH = 2). Thus, most lipids are
recovered without harmful effects on algae as diethyl ether is quickly evaporated from the
aqueous phase.
- an aliquot of the algae preculture in exponential growth, such that the initial cellular
concentration are about 5 000 cells. ml -1 fordiatoms and 100 cells.ml -1 fordinoflagellates.
Temperature is set at 18°C. Irradiance is 70µeinstein m. -2 s -1 with a 14 h.d -1 photoperiod.
All experiments are run under sterile conditions. Tests are performed until the end of
exponential growth.
The effects on algae growth are evaluated using in vivo fluorescence (on a Turner Designs
fluorometer) or by direct cells counting on hemacytometer. In vivo fluorescence is highly
correlated to cellular concentrations during the exponential growth of algae (Raimbault,
1982). This parameter is suitable to determine the general trends of pollutants effects on
phytoplankton.
Each point of the growth curves represents the mean value of three experiments.
Chemical analysis
Procedures used for sugars, amino acids and phosphates quantification are those described by Dreywood(1946), Mimura and Delmas (1983) and Strickland and Parsons (1972),
respectively.
622
Degradation of petroleum in continuous culture.
A crude oil (7.5 g.l -1 ) Asthart type is degraded continuously in fermentor containing
filtered sea water supplemented in PO4 (0.4 mM) at pH 8 and 30°C, with a dilution rate of
0.05 h - 1 . The fermentor is inoculated with a mixed bacterial culture which is composed of
8 strains isolated from sea water foams in hydrocarbons polluted areas. In such experimental conditions, the percentage of degraded crude oil is 83 %. The used medium is
centrifuged at 15 000 rpm for 15 mn. Remaining bacteria and most of residual petroleum
are removed from the used medium through 0.2 pm Millipore membrane before its
introduction in the culture medium of the test algae.
Test algae
Both algae are common species from the Gulf of Marseille : Phaeodactylum tricornutum
has been chosen because much is known about its physiology and its growth, even with
respect to hydrocarbon experiments (Lacaze and Villedon de Naide, 1976) ; (Lacaze,
1978 ; Kusk, 1981 a,b) : Prorocentrum micans is a Dinoflagellate often found in surface
slicks and possibly in connection with high lipid level in surface water (Kattner and
Brockmann, 1978 ; Cassie, 1981 ; De Souza Lima, 1981). Its growth is slower than that of
P. tricornutum.
Culture medium of algae
Cultures are grown in 40 ml test tubes closed with cotton plugs allowing volatiles to
escape. Tubes are successively filled up with :
- nutrients (Antia and Cheng algae medium)
- various percentages of the fermentor used medium (adjusted to 100 % in filtered sea
water). When required, lipids have been extracted from the used medium. This allows
lipids, lipid free used medium and untreated used medium to be tested separately on
phytoplankton. Each test is carried out with suitable control. The lipids are extracted
using diethyl ether after acidification of the medium (pH = 2). Thus, most lipids are
recovered without harmful effects on algae as diethyl ether is quickly evaporated from the
aqueous phase.
- an aliquot of the algae preculture in exponential growth, such that the initial cellular
concentration are about 5 000 cells. ml -1 fordiatoms and 100 cells.ml -1 fordinoflagellates.
Temperature is set at 18°C. Irradiance is 70µeinstein m. -2 s -1 with a 14 h.d -1 photoperiod.
All experiments are run under sterile conditions. Tests are performed until the end of
exponential growth.
The effects on algae growth are evaluated using in vivo fluorescence (on a Turner Designs
fluorometer) or by direct cells counting on hemacytometer. In vivo fluorescence is highly
correlated to cellular concentrations during the exponential growth of algae (Raimbault,
1982). This parameter is suitable to determine the general trends of pollutants effects on
phytoplankton.
Each point of the growth curves represents the mean value of three experiments.
Chemical analysis
Procedures used for sugars, amino acids and phosphates quantification are those described by Dreywood(1946), Mimura and Delmas (1983) and Strickland and Parsons (1972),
respectively.
622