to be no experimental data in the literature that can be used a priori to refute the existence
of highly adapted forms of microbial life in seawater at 250°C and 265 atm pressure
(Yanagawa and Kojima 1985). In fact, evidence is accumulating in support of our
findings, as discussed below.
Nevertheless, the conclusion that bacteria grew at temperatures of at least 250°C so
thoroughly questioned «conventional wisdom» (Walsby 1983) that some rejected our
work outright. Trent, Chastain, and Yayanos (1984), working with our culturing medium
but without vent samples or thermophilic bacteria, concluded that the evidence we
presented for bacterial growth at 250°C and 265 atm was based on «artifacts produced in
the medium and contaminants introduced during sample processing» and, therefore,
should not be «seriously considered supportive evidence for the existence of black smoker
bacteria». Their claims to have obtained results «nearly identical» to ours, using the same
experimental and analytical procedures but no bacterial inoculum, are misleading. Both
their methods and results differed from ours. For example, we stained samples with the
DNA-specific stain DAPI for 5 min, according to standard microbiological procedures,
while they stained for 1-3 h. We measured a linear increase in DAPI-staining «particles»,
while they reported a step-function increase immediately upon heating and erratic numbers thereafter. Our starting medium, freshly prepared and autoclaved, contained < 1 µg
total bound amino acids ml -1 (Deming 1984), while theirs contained 23µg ml -1 , primarily
in the form of serine, glutamic acid, glycine, and aspartic acid, the four classic contaminants in amino acids chromatography. At 250°C, we measured a linear increase (n=4;
r = .991), representing a doubling time of one hour, in the total amino acid content of
particulate protein (as well as in concentrations of each of the standardly-detected 15
amino acids and 5 unknown chromatographic peaks) from 1.6 to 145 µg ml -1 (Baross,
Deming, and Becker 1984), while they measured the aformentioned 23 µg ml -1 before
heating and 14µg ml -1 of the same 4 contaminant amino acids after heating. We photographed by transmission electron microscopy hundreds of intact bacteria in a sample
incubated 6 h at 250°C and 265 atm, many of which were unique in their resemblance to
known thermophiles (Stetter 1982), while Trent, Chastain, and Yayanos detected isolated
bacteria present as contaminants in their TEM embedding agar.
White (1984) rejected the existence of black smoker bacteria at 250°C and 265 atm on the
basis of his experiments showing the thermolability of various biomolecules from nonthermophilic organisms in glass testubes at 250°C and 39 atm vapor pressure. As discussed by Yanagawa and Kojima (1985), such experiments are irrelevant to studies of novel,
extremely thermophilic bacteria from the deep sea at their in situ hydrostatic pressure of
265 atm. Furthermore, White misinterpreted the micrographs published by Trent, Chastain, and Yayanos (1984), and misused those of Fox (1965), to argue that bacteria in our
micrographs were non-living materials condensed from soluble proteins and nucleic
acids. The levels of protein and nucleic acids used by Fox (and White) to generate
bacteria-like structures at high temperature were not present in our medium and the
similar structures of Trent, Chastain, and Yayanos (1984), by their own analysis, were
bacterial contaminants unrelated to experimentation at high temperature and pressure.
The issue of bacterial growth at 250°C will not be settled until we or others reproduce the
original growth studies (to this end, a modified version of the hydrothermal system used
in those experiments has recently been installed at the Chesapeake Bay Institute), but the
stability of some amino acids and peptide bonds at 250°C and 265 atm pressure has
recently been confirmed (Yanagawa and Kojima 1985), as well as the existence of
extremely thermophilic bacteria at smoker sites. While Trent, Chastain, Yayanos, and
White were developing what they published as refutations of our data, other investigators
327
of highly adapted forms of microbial life in seawater at 250°C and 265 atm pressure
(Yanagawa and Kojima 1985). In fact, evidence is accumulating in support of our
findings, as discussed below.
Nevertheless, the conclusion that bacteria grew at temperatures of at least 250°C so
thoroughly questioned «conventional wisdom» (Walsby 1983) that some rejected our
work outright. Trent, Chastain, and Yayanos (1984), working with our culturing medium
but without vent samples or thermophilic bacteria, concluded that the evidence we
presented for bacterial growth at 250°C and 265 atm was based on «artifacts produced in
the medium and contaminants introduced during sample processing» and, therefore,
should not be «seriously considered supportive evidence for the existence of black smoker
bacteria». Their claims to have obtained results «nearly identical» to ours, using the same
experimental and analytical procedures but no bacterial inoculum, are misleading. Both
their methods and results differed from ours. For example, we stained samples with the
DNA-specific stain DAPI for 5 min, according to standard microbiological procedures,
while they stained for 1-3 h. We measured a linear increase in DAPI-staining «particles»,
while they reported a step-function increase immediately upon heating and erratic numbers thereafter. Our starting medium, freshly prepared and autoclaved, contained < 1 µg
total bound amino acids ml -1 (Deming 1984), while theirs contained 23µg ml -1 , primarily
in the form of serine, glutamic acid, glycine, and aspartic acid, the four classic contaminants in amino acids chromatography. At 250°C, we measured a linear increase (n=4;
r = .991), representing a doubling time of one hour, in the total amino acid content of
particulate protein (as well as in concentrations of each of the standardly-detected 15
amino acids and 5 unknown chromatographic peaks) from 1.6 to 145 µg ml -1 (Baross,
Deming, and Becker 1984), while they measured the aformentioned 23 µg ml -1 before
heating and 14µg ml -1 of the same 4 contaminant amino acids after heating. We photographed by transmission electron microscopy hundreds of intact bacteria in a sample
incubated 6 h at 250°C and 265 atm, many of which were unique in their resemblance to
known thermophiles (Stetter 1982), while Trent, Chastain, and Yayanos detected isolated
bacteria present as contaminants in their TEM embedding agar.
White (1984) rejected the existence of black smoker bacteria at 250°C and 265 atm on the
basis of his experiments showing the thermolability of various biomolecules from nonthermophilic organisms in glass testubes at 250°C and 39 atm vapor pressure. As discussed by Yanagawa and Kojima (1985), such experiments are irrelevant to studies of novel,
extremely thermophilic bacteria from the deep sea at their in situ hydrostatic pressure of
265 atm. Furthermore, White misinterpreted the micrographs published by Trent, Chastain, and Yayanos (1984), and misused those of Fox (1965), to argue that bacteria in our
micrographs were non-living materials condensed from soluble proteins and nucleic
acids. The levels of protein and nucleic acids used by Fox (and White) to generate
bacteria-like structures at high temperature were not present in our medium and the
similar structures of Trent, Chastain, and Yayanos (1984), by their own analysis, were
bacterial contaminants unrelated to experimentation at high temperature and pressure.
The issue of bacterial growth at 250°C will not be settled until we or others reproduce the
original growth studies (to this end, a modified version of the hydrothermal system used
in those experiments has recently been installed at the Chesapeake Bay Institute), but the
stability of some amino acids and peptide bonds at 250°C and 265 atm pressure has
recently been confirmed (Yanagawa and Kojima 1985), as well as the existence of
extremely thermophilic bacteria at smoker sites. While Trent, Chastain, Yayanos, and
White were developing what they published as refutations of our data, other investigators
327