returned to the vents and collected black-smoker samples to examine them firsthand for
the presence or absence of extremely thermophilic bacteria. In each case, evidence was
obtained for the existence of extremely thermophilic bacteria in association with smoker
environments. Karl (1983) returned to 21°N on the Oasis Expedition, collected particles
in the plume of a 250-300°C smoker, and extracted from them 100 ng ATP per gram. This
represents a living biomass seyeral times greater than that found in overlying surface
waters and 10-100 times greater than surrounding, ambient deep-sea water. Karl et al.
(1984) reported rapid microbial activity in this same smoker sample at 90°C and 1 atm, the
most extreme conditions tested. From another sample of smoker fluid (unknown temperature), collected by Jannasch on the same expedition, Jones et al (1983) isolated the most
thermophilic methanogen yet discovered, Methanococcus jannaschii. From venting
fluids (unknown temperature) collected on a more recent ALVIN expedition, Jannasch
(1985) and Belkin and Jannasch (1985) also isolated extremely thermophilic, sulfurrespiring archaebacteria.
In March 1984, I joined the Byocyarise Expedition, organized by Daniel Desbruyères
(IFREMER, Brest, France), to 13°N along the East Pacific Rise. Using the submersible
“Cyana”, two samples of black fluid exiting from one of the multiple conduits at the apex
of a 17-m high smoker were collected in titanium water samplers pre-rinsed with acid and
.2 µm-filtered distilled water. Difficulties in positioning the entry pipe of the sampler into
the exiting conduit of this unusually tall smoker resulted in the collection of hydrothermal
fluid mixed with ambient seawater. The second sampling effort was similarly plagued but,
visually, appeared to have collected a purer sample of the black fluid (although a pH of 5
was measured for both samples after retrieval). Similar difficulties with manipulations of
the temperature probe gave maximum recordings of 138°C in the plume, where temperatures up to 280°C had been measured at the same point during earlier dives. After water
collection, a 15 cm 3 rock that included an exiting conduit for the black fluid was removed
from the smoker, using the pincer of the “Cyana” arm. Finally, a third titanium water
sampler was filled several hundred meters from the smoker as an ambient seawater
control.
Aboard the Research Vessel “Nadir”, the water samples were expressed from the syringelike samplers into sterile containers. A portion of each sample was split into 4.5-ml
aliquots, each of which was then spiked with 0.5 ΜC+ 3 H-thymidine (1 Ci/mMol ; CEA,
France) and loaded into a sterile glass syringe. The syringes were returned to vent pressure
(265 atm) in pressure vessels (Tem-Pres Division of Leco Corporation, P.O. Box 390,
Blanchard St. Extension, Bellefonte, PA 16823 USA) and incubated 10 h at 2, 55, and
98°C (the hottest temperature that could be reached in the available shipboard oven).
After decompression, samples were extracted in cold 5% tricarboxylic acid (TCA),
vacuum-filtered onto .2 um filters, and rinsed with twice the volume of TCA. Radioactivity in the particulate fraction was assayed by scintillation counting (counting efficiency
was 80.4 %)) and compared to duplicate samples extracted and filtered immediately after
addition of label. Dpm ml" were corrected against killed controls using HgCl 2 . The results
of this experiment indicated thermophilic microbial activity associated with blacksmoker effluent but not with ambient seawater. Although no increases in dpm ml - were
measured in the first hot water sample under any of the test conditions, dpm ml - in the
second one doubled in 10 h at 55 and 98°C with no increase measured at 2°C. Dpm ml - in
the ambient seawater control increased 6-fold at 2°C, but decreased about 50 % at 55 and
98°C.
In addition to thymidine uptake studies, a variety of different media compositions,
selective for chemolithotrophic bacteria or devised for other known thermophiles, were
inoculated with portions of the hot water samples and sealed in serum bottles or tubes.
328
the presence or absence of extremely thermophilic bacteria. In each case, evidence was
obtained for the existence of extremely thermophilic bacteria in association with smoker
environments. Karl (1983) returned to 21°N on the Oasis Expedition, collected particles
in the plume of a 250-300°C smoker, and extracted from them 100 ng ATP per gram. This
represents a living biomass seyeral times greater than that found in overlying surface
waters and 10-100 times greater than surrounding, ambient deep-sea water. Karl et al.
(1984) reported rapid microbial activity in this same smoker sample at 90°C and 1 atm, the
most extreme conditions tested. From another sample of smoker fluid (unknown temperature), collected by Jannasch on the same expedition, Jones et al (1983) isolated the most
thermophilic methanogen yet discovered, Methanococcus jannaschii. From venting
fluids (unknown temperature) collected on a more recent ALVIN expedition, Jannasch
(1985) and Belkin and Jannasch (1985) also isolated extremely thermophilic, sulfurrespiring archaebacteria.
In March 1984, I joined the Byocyarise Expedition, organized by Daniel Desbruyères
(IFREMER, Brest, France), to 13°N along the East Pacific Rise. Using the submersible
“Cyana”, two samples of black fluid exiting from one of the multiple conduits at the apex
of a 17-m high smoker were collected in titanium water samplers pre-rinsed with acid and
.2 µm-filtered distilled water. Difficulties in positioning the entry pipe of the sampler into
the exiting conduit of this unusually tall smoker resulted in the collection of hydrothermal
fluid mixed with ambient seawater. The second sampling effort was similarly plagued but,
visually, appeared to have collected a purer sample of the black fluid (although a pH of 5
was measured for both samples after retrieval). Similar difficulties with manipulations of
the temperature probe gave maximum recordings of 138°C in the plume, where temperatures up to 280°C had been measured at the same point during earlier dives. After water
collection, a 15 cm 3 rock that included an exiting conduit for the black fluid was removed
from the smoker, using the pincer of the “Cyana” arm. Finally, a third titanium water
sampler was filled several hundred meters from the smoker as an ambient seawater
control.
Aboard the Research Vessel “Nadir”, the water samples were expressed from the syringelike samplers into sterile containers. A portion of each sample was split into 4.5-ml
aliquots, each of which was then spiked with 0.5 ΜC+ 3 H-thymidine (1 Ci/mMol ; CEA,
France) and loaded into a sterile glass syringe. The syringes were returned to vent pressure
(265 atm) in pressure vessels (Tem-Pres Division of Leco Corporation, P.O. Box 390,
Blanchard St. Extension, Bellefonte, PA 16823 USA) and incubated 10 h at 2, 55, and
98°C (the hottest temperature that could be reached in the available shipboard oven).
After decompression, samples were extracted in cold 5% tricarboxylic acid (TCA),
vacuum-filtered onto .2 um filters, and rinsed with twice the volume of TCA. Radioactivity in the particulate fraction was assayed by scintillation counting (counting efficiency
was 80.4 %)) and compared to duplicate samples extracted and filtered immediately after
addition of label. Dpm ml" were corrected against killed controls using HgCl 2 . The results
of this experiment indicated thermophilic microbial activity associated with blacksmoker effluent but not with ambient seawater. Although no increases in dpm ml - were
measured in the first hot water sample under any of the test conditions, dpm ml - in the
second one doubled in 10 h at 55 and 98°C with no increase measured at 2°C. Dpm ml - in
the ambient seawater control increased 6-fold at 2°C, but decreased about 50 % at 55 and
98°C.
In addition to thymidine uptake studies, a variety of different media compositions,
selective for chemolithotrophic bacteria or devised for other known thermophiles, were
inoculated with portions of the hot water samples and sealed in serum bottles or tubes.
328