cyanobacterial mat on the island of Mellum, southern North Sea (Stal and Krumbein,
1981; Stal et al, 1984c). Both strains are typical marine cyanobacteria: no growth was
observed in freshwater medium. Oscillatoria sp. strain 23, a filamentous, non-heterocystous cyanobacterium, fixes molecular nitrogen even under complete aerobic conditions (Stal and Krumbein, 1981). This organism shows good growth in media depleted of
a source of fixed nitrogen. Both organisms were grown in artificial seawater medium
ASN III (Rippka et al, 1979). Oscillatoria was also grown on the combined nitrogen free
variant of this medium (ASN III
0 ) for some experiments, to induce nitrogenase. Organisms were grown in a Gallenkamp (London) illuminated shaking incubator at 20°C with
1300 and 2500 lux light intensity for Oscillatoria and Microcoleus respectively.
Anaerobic dark incubation
Cultures were centrifuged at 10,000 g and washed twice in a buffered medium containing
25 g.l -1 NaCl and 10mM TES (N-Tris (hydroxymethyl)-methyl-2-aminoethane-sulfonic
acid), pH 7.9. The filaments were then suspended in this medium. Ten ml of suspension was
filled in a 12 ml tube and 1 ml of a suspension of elemental sulfur or 1 ml H
2
O was added.
The tubes were wrapped in alumina foil and bubbled with oxygen free helium for 5
minutes. The tubes were closed with a serum stopper and incubated at room temperature.
After 6, 12, 24 and 54 hours of incubation tubes were centrifuged and pellet and
supernatant analyzed for chlorophyll a, glycogen, H 2 S and lactate. The tubes contained
60 pg, 200 pg and 50 µg chlorophyll a for M. chthonoplastes, Oscillatoria grown on
ASN III and Oscillatoria grown on ASN III °, respectively. For acetylene reduction measurements, 10 ml of Oscillatoria were transferred into a 40 ml serum bottle. The bottles
were wrapped in alumina foil and bubbled with helium. After closing the bottle, 15% of
acetylene was injected, using a gastight syringe.
Nitrogenase activity
Nitrogenase activity was measured as acetylene reduction (Stewart et al, 1967). Acetylene
and ethylene were determined gas chromatographically as described previously (Stal and
Krumbein, 1981; Stal et al, 1984b).
Preparation of fine dispersed elemental sulfur
A suspension of finely dispersed elemental sulfur was prepared according to Roy and
Trudinger (1970). This suspension contained approximatively 1.3 mg S.ml -1 as was
determined by the method of Stal et al. (1984a).
Determination of sulfide
Sulfide was determined colorimetrically after Pachmayr (Trüper and Schlegel, 1964).
Determination of L-lactate
L-lactate was determined enzymatically using the Boehringer Test-Combination (Boehringer, W-Germany) (Noll, 1974).
Determination of glycogen
Glycogen was determined as glucose after hydrolyzing the cells in 10 ml 2 N HCl for 6
hours at 100°C. The hydrolysate was neutralized with 10 ml 2 N NaOH and 16 ml 200 mM
phosphate buffer, pH 7.0 was added. The volume was adjusted to 50 ml with distilled
water. Glucose was determined by the GOD-Perid-Method, Boehringer Test-Com303
1981; Stal et al, 1984c). Both strains are typical marine cyanobacteria: no growth was
observed in freshwater medium. Oscillatoria sp. strain 23, a filamentous, non-heterocystous cyanobacterium, fixes molecular nitrogen even under complete aerobic conditions (Stal and Krumbein, 1981). This organism shows good growth in media depleted of
a source of fixed nitrogen. Both organisms were grown in artificial seawater medium
ASN III (Rippka et al, 1979). Oscillatoria was also grown on the combined nitrogen free
variant of this medium (ASN III
0 ) for some experiments, to induce nitrogenase. Organisms were grown in a Gallenkamp (London) illuminated shaking incubator at 20°C with
1300 and 2500 lux light intensity for Oscillatoria and Microcoleus respectively.
Anaerobic dark incubation
Cultures were centrifuged at 10,000 g and washed twice in a buffered medium containing
25 g.l -1 NaCl and 10mM TES (N-Tris (hydroxymethyl)-methyl-2-aminoethane-sulfonic
acid), pH 7.9. The filaments were then suspended in this medium. Ten ml of suspension was
filled in a 12 ml tube and 1 ml of a suspension of elemental sulfur or 1 ml H
2
O was added.
The tubes were wrapped in alumina foil and bubbled with oxygen free helium for 5
minutes. The tubes were closed with a serum stopper and incubated at room temperature.
After 6, 12, 24 and 54 hours of incubation tubes were centrifuged and pellet and
supernatant analyzed for chlorophyll a, glycogen, H 2 S and lactate. The tubes contained
60 pg, 200 pg and 50 µg chlorophyll a for M. chthonoplastes, Oscillatoria grown on
ASN III and Oscillatoria grown on ASN III °, respectively. For acetylene reduction measurements, 10 ml of Oscillatoria were transferred into a 40 ml serum bottle. The bottles
were wrapped in alumina foil and bubbled with helium. After closing the bottle, 15% of
acetylene was injected, using a gastight syringe.
Nitrogenase activity
Nitrogenase activity was measured as acetylene reduction (Stewart et al, 1967). Acetylene
and ethylene were determined gas chromatographically as described previously (Stal and
Krumbein, 1981; Stal et al, 1984b).
Preparation of fine dispersed elemental sulfur
A suspension of finely dispersed elemental sulfur was prepared according to Roy and
Trudinger (1970). This suspension contained approximatively 1.3 mg S.ml -1 as was
determined by the method of Stal et al. (1984a).
Determination of sulfide
Sulfide was determined colorimetrically after Pachmayr (Trüper and Schlegel, 1964).
Determination of L-lactate
L-lactate was determined enzymatically using the Boehringer Test-Combination (Boehringer, W-Germany) (Noll, 1974).
Determination of glycogen
Glycogen was determined as glucose after hydrolyzing the cells in 10 ml 2 N HCl for 6
hours at 100°C. The hydrolysate was neutralized with 10 ml 2 N NaOH and 16 ml 200 mM
phosphate buffer, pH 7.0 was added. The volume was adjusted to 50 ml with distilled
water. Glucose was determined by the GOD-Perid-Method, Boehringer Test-Com303