Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2920Blackwell Publishing Ltd, 200357583591Original ArticleMetabolism of organics in hydrothermal sedimentJ. M. Tor, J. P. Amend and D. R. Lovley
Environmental Microbiology (2003) 5(7), 583–591
Metabolism of organic compounds in anaerobic,
hydrothermal sulphate-reducing marine sediments
Jason M. Tor,1† Jan P. Amend2 and Derek R. Lovley1*
Department of Microbiology, University of
Massachusetts, Amherst, MA 01003, USA.
2
Department of Earth and Planetary Sciences,
Washington University, St. Louis, MO 63130,USA.
Introduction
1
Summary
Previous studies of hot (>80∞C) microbial ecosystems
have primarily relied on the study of pure cultures or
analysis of 16S rDNA sequences. In order to gain
more information on anaerobic metabolism by natural
communities in hot environments, sediments were
collected from a shallow marine hydrothermal vent
system in Baia di Levante, Vulcano, Italy and incubated under strict anaerobic conditions at 90∞∞C. Sulphate reduction was the predominant terminal
electron-accepting process in the sediments. The
addition of molybdate inhibited sulphate reduction in
the sediments and resulted in a linear accumulation
of acetate and hydrogen over time. [U-14C]- acetate
was completely oxidized to 14CO2, and the addition of
molybdate inhibited 14CO2 production by 60%. [U-14C]glucose was oxidized to 14CO2, and this was inhibited
when molybdate was added. When the pool sizes of
short-chain fatty acids were artificially increased,
radiolabel from [U-14C]-glucose accumulated in the
acetate pool. L-[U-14C]-glutamate, [ring-14C]-benzoate
and [U-14C]-palmitate were also anaerobically oxidized
to 14CO2 in the sediments, but molybdate had little
effect on the oxidation of these compounds. These
results demonstrate that natural microbial communities living in a hot, microbial ecosystem can oxidize
acetate and a range of other organic electron donors
under sulphate-reducing conditions and suggest that
acetate is an important extracellular intermediate in
the anaerobic degradation of organic matter in hot
microbial ecosystems.
Received 23 September, 2002; revised 15 January, 2003; accepted
27 January, 2003. *For correspondence. E-mail dlovley@microbio.
umass.edu, Tel. (+1) 413 545 9651; Fax (+1) 413 545 1578. †Current
address: School of Natural Science, Hampshire College, Amherst,
MA 01002, USA.
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd
Although there is substantial interest in hot (i.e. >80∞C),
anaerobic, microbial ecosystems, previous models for
microbial metabolism of organic matter in these environments have been based on the results of studies with pure
cultures (Bonch-Osmolovskaya, 1994; Kelly and Adams,
1994; Slobodkin et al., 1999). This contrasts with studies
on anaerobic metabolism in more temperate sedimentary
environments in which the metabolism of complex microbial communities has been directly studied (for reviews
see: Lovley and Chapelle, 1995; Conrad, 1996). In temperate anaerobic sediments, fermentative and respiratory
microorganisms co-operate to completely oxidize complex
organic matter to carbon dioxide with Fe(III), Mn(IV), SO42–,
and CO2 serving as the terminal electron acceptor. A key
step in this metabolism is the oxidation of acetate coupled
to the reduction of the terminal electron acceptors,
because acetate is the most prevalent organic fermentation product.
The majority of hyperthermophiles in pure culture are
obligate heterotrophs, which preferentially ferment complex mixtures of polypeptides and/or carbohydrates (Kelly
and Adams, 1994; Schroder et al., 1994; Schonheit and
Schafer, 1995; Kengen et al., 1996). The unproven ability
of hyperthermophiles to anaerobically oxidize acetate in
situ or in laboratory culture led to the suggestion that
acetate produced in hot microbial ecosystems from fermentation, or thermocatalysis of organic matter in hydrothermally altered sediments (Martens, 1990), needed to
diffuse into cooler environments in order to be metabolised (Slobodkin et al., 1999). However, subsequent studies documented that two hyperthermophiles, Ferroglobus
placidus and Geoglobus ahangari, could oxidize acetate
with Fe(III) serving as the electron acceptor (Tor et al.,
2001). Additional studies demonstrated that these
organisms could also oxidize various aromatic compounds (Tor and Lovley, 2001) and long-chain fatty acids
(Kashefi et al., 2002) with the reduction of Fe(III). This
suggests that a consortia of hyperthermophiles, consisting of fermentative microorganisms and acetate-, benzoate- and long-chain fatty acid-oxidizing Fe(III) reducers,
may be able to bring about the complete oxidation of
complex organic compounds in organic-rich hyperthermophilic environments (Lovley et al., 2003). However, such
an oxidation of complex organic matter coupled to Fe(III)
584 J. M. Tor, J. P. Amend and D. R. Lovley
reduction has not been documented in hot, anoxic
sediments.
The complete oxidation of acetate, aromatic compounds and long-chain fatty acids coupled to reduction of
other electron acceptors such as nitrate and sulphurspecies is also thermodynamically favourable under the
geochemical conditions found in many hydrothermal ecosystems (Amend and Shock, 2001). Although acetate oxidation coupled to the reduction of nitrate (Volkl et al.,
1993) or sulphite (Huber et al., 1997) has been proposed
for pure cultures, data directly demonstrating this were not
provided and subsequent studies refuted these claims
(Afshar et al., 1998; Tor et al., 2001). Information on
anaerobic metabolism of organic matter coupled to sulphate reduction is of particular interest due the availability
of high concentrations of sulphate in many hot marine
sediments. In pure culture studies, a strain of Archaeoglobus fulgidus was shown to completely oxidize lactate
when using sulphate as its terminal electron acceptor
(Beeder et al., 1994); however, earlier reports of lactate
utilization by A. fulgidus described fermentation of the
compound to acetate and CO2 (Zellner et al., 1989). Neither strain was shown to oxidize acetate coupled to sulphate reduction.
In order to evaluate the anaerobic metabolism of
organic matter in hot, microbial ecosystems, marine sediments from a shallow hydrothermal vent in Vulcano, Italy
were investigated. The results demonstrate that, like more
temperate anoxic marine sediments, sulphate reduction is
an important process for the anaerobic oxidation of fermentation intermediates, particularly acetate, and that cooperative activity between fermentative microorganisms
and sulphate reducers is important for the metabolism of
fermentable compounds, in these hot sediments.
mole of electrons per mole of Fe(II) produced it is apparent that sulphate reduction accounted for over 75-fold
more electron flow than Fe(III) reduction in these
sediments.
Effect of molybdate on sulphate reduction and metabolism
of fermentation intermediates
Molybdate inhibited sulphate reduction in the hyperthermophile Archaeoglobus profundus when added at
equimolar concentrations with sulphate, suggesting that,
as has been previously observed with mesophilic sulphate
reducers (Oremland and Capone, 1988), molybdate may
be an effective inhibitor of sulphate reduction in hyperthermophiles. Molybdate also inhibited sulphate reduction in
the complex microbial communities living in the hot marine
sediments from Vulcano when the incubations were
amended with acetate, glucose and H2 (Fig. 1).
When sulphate reduction in the sediments was inhibited
with molybdate, there was a linear accumulation of acetate (15.0 nmol g-1day-1) and hydrogen (0.48 pmol g-1
day-1) whereas there was no significant increase in
acetate and hydrogen in sediments until amended with
molybdate (Fig. 2). The acetate concentration in laboratory sediments before amendment with molybdate
remained steady at 0.48 mmol g-1 dry sediment, which is
equivalent to 1.9 mM. The volatile fatty acids in fluids from
numerous hydrothermal springs on Vulcano have been
Results
Sulphate reduction as the predominant terminal
electron accepting process
Sulphate reduction appeared to be the predominant terminal electron-accepting process in the sediments. Common terminal electron acceptors such as nitrate, sulphite
and thiosulphate were not detected in the laboratory incubations. S0 was present at almost 24 mmol per gram of dry
sediment, but was not depleted over time, suggesting that
it was not an important electron acceptor. Sulphate was
steadily depleted over time at a rate of 72 mM day-1 (equivalent to 23.8 nmol g-1 dry sediment day-1). There was
approximately 800 nmol HCl-extractable Fe(III) per gram
of sediment (dry), but Fe(II) only accumulated at a rate of
2.5 nmol Fe2+ g-1 dry sediment day-1. When it is considered that each mole of sulphate reduced consumes eight
moles of electrons and Fe(III) reduction consumes one
Fig. 1. Reduction of sulphate in sediments amended with acetate
(5 mM), glucose (5 mM), and H2:CO2 (80:20, v/v), incubated at 90∞C.
() Sediment without molybdate, () sediment with added molybdate
(25 mM). Data shown are means of three replicates. Error bars represent one standard deviation.
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 583–591
Metabolism of organics in hydrothermal sediment 585
Fig. 2. Accumulation of H2 () and acetate () in sediments
amended with molybdate (25 mM), molybdate addition indicated by
the arrow.
measured (Amend et al., 1998) and shown to contain
acetic acid concentrations in the order of 2.2–3.1 mM. The
addition of molybdate did not result in an accumulation
of methane, formate, propionate or butyrate in the
sediments.
Fate of 14C-labelled compounds
[U-14C]-acetate added to the sediments was completely
oxidized to 14CO2 in 24 h (Fig. 3). Molybdate inhibited the
oxidation of [U-14C]-acetate by about 60%. There was no
production of 14CH4 from [U-14C]-acetate in the presence
or absence of molybdate. There was no detectable accumulation of Fe(II) over the course of the radiolabel experiment (24 h). These results further suggest that sulphate
reduction was the predominant terminal electron accepting process in the sediments (Lovley, 1997).
[U-14C]-glucose was completely oxidized to 14CO2 in the
sediments within 24 h (Fig. 4). Molybdate greatly inhibited
glucose oxidation. When a mixture of short-chain fatty
acids, including acetate, propionate, butyrate and lactate
were added to the sediment, the production of 14CO2 was
diminished, with only 29% of the added 14C appearing as
14
CO2. 43% of the radiolabel was recovered in fatty acids
after 24 h, primarily in acetate (Fig. 5). There was no
production of 14CH4 from [U-14C]-glucose.
L-[U-14C]-glutamate added to sediments was partially
converted to 14CO2 with 37% of the added radiolabel
appearing as 14CO2 within 20 h and 42% after 6 days
(Fig. 6). Molybdate partially inhibited the production of
14
CO2 over 20 h, but had no inhibitory effect in later time
points. Addition of short-chain fatty acids to sediments
containing L-[U-14C]-glutamate resulted in the production
Fig. 3. Recovery of added [U-14C]-acetate as 14CO2 from sediments
amended with () [U-14C]-acetate (), [U-14C]-acetate and molybdate
(25 mM), or () [U-14C]-acetate in autoclaved sediments. Data shown
are means of three replicates. Error bars represent one standard
deviation.
of both 14C-labelled intermediates and 14CO2. Approximately 31% of the added 14C was converted to 14CO2, and
8% of the radiolabel was recovered in the organic acids
after 6 days. Acetate was the primary intermediate in the
fermentation of the L-[U-14C]-glutamate, accounting for
5% of the added 14C (Fig. 5). There was no production of
14
CH4 from L-[U-14C]-glutamate.
Fig. 4. Recovery of added [U-14C]-glucose as 14CO2 from sediments
amended with () [U-14C]-glucose (), [U-14C]-glucose and molybdate (25 mM) ,() [U-14C]-glucose in autoclaved sediments, or (▲)
14
C-glucose and short chain organic acids. Data shown are means of
three replicates. Error bars represent one standard deviation.
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 583–591
586 J. M. Tor, J. P. Amend and D. R. Lovley
13% of the added 14C, whereas acetate accounted for
1.4% of the added 14C (Fig. 5).
[U-14C]-palmitate added to the sediments was completely oxidized to 14CO2 within 30 h (Fig. 8). The addition
of molybdate only slightly inhibited production of 14CO2
and there was no production of 14CH4. Isotope trapping
studies were not performed with palmitate.
Discussion
Fig. 5. Accumulation of radioactivity added to sediments containing
short-chain organic acids. Data shown are means of three replicates.
Error bars represent one standard deviation.
[Ring-14C]-benzoate was slowly oxidized to 14CO2 with
66% of the added 14C accumulating as 14CO2 after 21 days
(Fig. 7). Molybdate did not significantly impact on the rate
of benzoate oxidation. No 14CH4 was produced. When a
mixture of short-chain organic acids was added to the
sediments, the production of 14CO2 was greatly inhibited
and 20% of the added radiolabel was recovered in the
fatty acid pool. Butyrate was the primary intermediate in
the fermentation of the [ring-14C]-benzoate, accounting for
Fig. 6. Recovery of added L-[U-14C]-glutamate as 14CO2 from sediments amended with () L-[U-14C]-glutamate, () L-[U-14C]glutamate and molybdate (25 mM) (), L-[U-14C]-glutamate in autoclaved sediments, or (▲) L-[U-14C]-glutamate and short chain organic
acids. Data shown are means of three replicates. Error bars represent
one standard deviation.
These results demonstrate that the microorganisms living
in hot, marine sediments have a significant potential for
the anaerobic oxidation of a variety of organic compounds. As detailed below, in some respects the metabolism of organic compounds in the Vulcano sediments is
similar to that found in anoxic marine sediments at more
moderate temperatures in which sulphate reduction is the
predominant terminal electron accepting process. For
example, sulphate-reducing microorganisms appear to be
involved in the metabolism of both acetate and hydrogen
produced by fermentative microorganisms and thus fermentative microorganisms and sulphate reducers cooperate to oxidize glucose.
Sulphate reduction and inhibition with molybdate
Sulphate served as a terminal electron acceptor for anaerobic metabolism in the Vulcano sediments as evidenced
Fig. 7. Recovery of added [ring-14C]-benzoate as 14CO2 from sediments amended with () [ring-14C]-benzoate, () [ring-14C]-benzoate
and molybdate (25 mM), () [ring-14C]-benzoate in autoclaved sediments, or (▲) [ring-14C]-benzoate and short chain organic acids. Data
shown are means of three replicates. Error bars represent one standard deviation.
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 583–591
Metabolism of organics in hydrothermal sediment 587
Fig. 8. Recovery of added [U-14C]-palmitate as 14CO2 from sediments
amended with () [U-14C]-palmitate, () [U-14C]-palmitate and molybdate (25 mM), or () [U-14C]-palmitate in autoclaved sediments. Data
shown are means of three replicates. Error bars represent one standard deviation.
from the steady loss of sulphate over time in sediments
incubated under anoxic conditions. The rate of sulphate
depletion in the Vulcano sediments (72 mM day-1;
23.8 nmol g-1 dry sediment day-1) is similar to rates of
sulphate reduction in hot marine sediments from Guaymas Basin of 19–62 mM day-1 (Jorgensen et al., 1992)
and 30–75 nmol g-1day-1 (Elsgaard et al., 1994), each of
which were estimated with the 35SO4- technique.
The addition of molybdate inhibited sulphate depletion
in the Vulcano sediments. This is consistent with the wellknown ability of molybdate to serve as an inhibitor for
sulphate reduction in anoxic marine sediments at more
moderate temperatures (Oremland and Capone, 1988).
Molybdate also inhibited sulphate reduction in cultures of
A. profundus. These results suggest that molybdate is a
useful inhibitor for studying the activity of sulphatereducing microorganisms in hot, marine sediments.
mentation products when terminal electron accepting
processes are inhibited can provide insights into the role
of the microorganisms involved in the terminal process
and gives an indication of the importance of potential
fermentation intermediates in carbon and electron flow
(Sorensen et al., 1981; Lovley and Klug, 1982; Krumbock
and Conrad, 1991; Chidthaisong et al., 1999; Chidthaisong and Conrad, 2000). Thus, the results suggest that
acetate and hydrogen were intermediates in the anaerobic degradation of organic matter in the Vulcano sediments and that sulphate-reducing microorganisms were
responsible for acetate and hydrogen metabolism.
The rate of acetate accumulation in molybdateamended sediments suggested that acetate may account
for as much as 63% of the sulphate reduction in
unamended sediments. The accumulation of hydrogen
suggested that hydrogen accounted for less than 1% of
the sulphate reduction in the sediments, but this may be
an underestimate because increased hydrogen concentrations may have inhibited hydrogen production. Other
fermentation acids such as propionate and butyrate,
which have been found to accumulate as minor intermediates when terminal metabolism is inhibited in more temperate sediments (Sorensen et al., 1981; Christensen,
1984; Krumbock and Conrad, 1991; Kusel and Dorsch,
2000), did not accumulate to detectable levels in the Vulcano sediments.
Sulphate-reducing microorganisms were largely
responsible for acetate metabolism in the Vulcano sediments, which was further suggested by the observation
that the addition of molybdate significantly inhibited the
metabolism of [U-14C]-acetate. However, there appeared
to be processes other than sulphate reduction involved in
acetate metabolism because molybdate did not completely inhibit the oxidation of [U-14C]-acetate. There was
no conversion of [U-14C]-acetate to 14CH4, demonstrating
that there was not a metabolically active population of
acetate-utilizing methanogens in the sediments. The low
rates of Fe(III) reduction and the lack of S∞ reduction in
the sediments suggested that Fe(III) and S∞ reduction
could not be important processes for acetate oxidation in
situ, but the possibility remains that once sulphate reduction was inhibited then some acetate was oxidized with
Fe(III) and/or S∞ reduction.
Role of sulphate reducers in acetate and hydrogen
metabolism
Glucose metabolism
Sulphate-reducing microorganisms appear to play an
important role in the metabolism of acetate and hydrogen
in the Vulcano sediments. When sulphate reduction was
inhibited with molybdate, there was a linear accumulation
of acetate and hydrogen. This response was similar to that
observed in marine sediments at more moderate temperatures (Sorensen et al., 1981). The accumulation of fer-
The results suggest that glucose, a common model for the
metabolism of simple sugars in sediments, was fermented
rather than being directly oxidized to carbon dioxide. The
metabolism of [U-14C]-glucose in anaerobic, mesophilic
sediments is complex (King and Klug, 1982), but the accumulation of 14C-metabolic products gives a qualitative indication of how glucose is metabolised (King and Klug,
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 583–591
588 J. M. Tor, J. P. Amend and D. R. Lovley
1982; Lovley and Philips, 1989; Sawyer and King, 1993).
The finding that increasing the pool sizes of fermentation
acids in the sediments trapped the radiolabel from [U-14C]glucose metabolism and inhibited production of 14CO2
demonstrated that glucose was primarily being fermented
in the sediments. This conclusion is consistent with the
initial lag phase in production of 14CO2 from [U-14C]-glucose in the sediments that were not amended with fatty
acids, as the diversion of the radiolabel into fermentation
acids would delay its conversion to 14CO2. The accumulation of most of the radiolabel in the acetate pool in the
sediments amended with fatty acids suggested that most
of the glucose was fermented to acetate, hydrogen, and
carbon dioxide. Thus, the inhibition of 14CO2 production
from [U-14C]-glucose with molybdate can probably be
attributed primarily to an inhibition of acetate oxidation
rather than a direct impact on the metabolism of glucose.
Some hyperthermophiles, such as Thermoproteus
tenax and Pyrobaculum islandicum, are capable of completely oxidizing fermentable substrates, including glucose, directly to CO2 with sulphur or thiosulphate as
electron acceptors without the production of short-chain
fatty acids (Selig and Schonheit, 1994). However,
hyperthermophilic sulphate reducers capable of completely oxidizing sugars to carbon dioxide with sulphate as
the electron acceptor have not been described. There are
many pure cultures of hyperthermophilic Archaea and
Bacteria that ferment glucose to primarily acetate,
hydrogen and carbon dioxide production (Selig et al.,
1997).
Metabolism of glutamate, benzoate and palmitate
Dissolved free amino acids in vent fluids and porewaters
comprise a potential source of both organic carbon and
nitrogen for heterotrophic assimilation, and represent
important intermediates in organic matter remineralization
in hot environments (Haberstroh and Karl, 1989). In the
biogenic zone of hydrothermally impacted sediments in
Guaymas Basin elevated concentrations of dissolved free
amino acids, including glutamate (10–50 mM in near surface sediments), have been observed and attributed to in
situ production of microbial biomass (Haberstroh and Karl,
1989). Unlike glucose, only a small fraction of the added
L-[U-14C]-glutamate was trapped in fermentation acids
when their concentration was increased in the Vulcano
sediments, indicating fermentation was a minor process
in the metabolism of the radiolabelled glutamate. As only
about a third of the added L-[U-14C]-glutamate was recovered as 14CO2, even after 6 days of incubation, it is likely
that much of the glutamate was assimilated as cell carbon,
but this was not verified. The lack of effect of molybdate
on glutamate oxidation suggests that sulphate reducing
microorganisms are not important in glutamate oxidation,
or that other microorganisms can metabolise glutamate
when sulphate reducers are inhibited.
Aromatic compounds are a potentially important component of organic carbon in hot (>80∞C) anaerobic ecosystems, such as petroleum reservoirs (Magot et al.,
2000), organic-rich sediments in marine hydrothermal
zones (Goetz and Jannasch, 1993) and some marine
hydrothermal vents (Simoneit and Lonsdale, 1982). The
complete oxidation of the several aromatic compounds,
including benzoate, with Fe(III) serving as the electron
acceptor was recently reported in the hyperthermophile
Ferroglobus placidus (Tor and Lovley, 2001), an isolate
from the shallow marine hydrothermal system on Vulcano,
Italy (Hafenbradl et al., 1996). That finding suggested that
the potential existed for at least some aromatic compounds to be oxidized anaerobically in the hot sediments.
The studies with [ring-14C]-benzoate demonstrate that
aromatic compounds can be anaerobically oxidized in the
Vulcano sediments. However, the lack of effect of molybdate on benzoate oxidation suggests that sulphate reducing microorganisms are not important in benzoate
oxidation, or that other microorganisms can metabolize
benzoate when sulphate reducers are inhibited. Butyrate
was the primary intermediate in the fermentation of the
[ring-14C]-benzoate. This contrasts with benzoate fermentation in mesophilic environments in which acetate and
carbon dioxide are the primary products of benzoate fermentation, with only minor accumulations of butyrate
(Heider and Fuchs, 1997).
Long-chain fatty acids represent another component of
the complex assemblage of organic matter found in many
sedimentary environments. The complete oxidation of
the long-chain fatty acids, palmitate and stearate, was
recently reported in the hyperthermophile Geoglobus
ahangari, an isolate from Guaymas Basin utilizing Fe(III)
as an electron acceptor (Kashefi et al., 2002). In this
study, [U-14C]-palmitate was completely oxidized to 14CO2
within 30 h. Molybdate had no effect on palmitate oxidation, suggesting that a respiratory process other than sulphate reduction may be involved in the metabolism of the
palmitate.
Conclusions
These results demonstrate that acetate is an important
intermediate in the anaerobic degradation of organic matter in the hot Vulcano sediments and that sulphate-reducing microorganisms play an important role in acetate
oxidation in the sediments. These results provide further
support for the concept that acetate can be anaerobically
oxidized within hot microbial ecosystems (Tor et al., 2001;
Lovley et al., 2003), rather than diffusing into cooler environments, as has previously been proposed (Slobodkin
et al., 1999). This emphasises the importance of directly
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 583–591
Metabolism of organics in hydrothermal sediment 589
examining microbial metabolism in natural microbial communities rather than making extrapolations from pure culture studies. The metabolism of glucose via fermentation
primarily to acetate followed by acetate oxidation coupled
to sulphate reduction is similar to the pathway for glucose
metabolism in anoxic marine sediments of more moderate
temperature. This suggests that key concepts about
organic matter metabolism in cooler sediments may also
apply to anaerobic metabolism in hot, microbial ecosystems as well. The finding that molybdate is an effective
inhibitor of sulphate reduction in hot marine sediments
provides a useful tool for studying anaerobic metabolism
in these environments. Clearly, further investigation of
metabolism in the Vulcano sediments is warranted in order
to identify the electron acceptors other than sulphate that
appear to be involved in anaerobic oxidation of organic
compounds and to further define the range of organic
compounds which can be anaerobically metabolised in
these sediments.
Experimental procedures
Culture of Archaeoglobus profundus
Archaeoglobus profundus (DSM 5631) was obtained from the
type culture collection of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. The culture was grown as described previously
(Burggraf et al., 1990; Tor and Lovley, 2001) with sulphate
(10 mM) as the electron acceptor. Molybdate (10 mM) was
added to inhibit sulphate reduction.
Study site
Sediment from a marine hydrothermal vent was investigated.
Marine sediment (10–20 cm depth) was collected from a
shallow (30 cm) hydrothermal vent in Baia di Levante, Vulcano, Italy. The fluids at this depth were about 90∞C, pH 5.5,
and composed primarily of hydrothermally altered beach
sands. The sediment was stored in a canning jar and immediately incubated at 80∞C overnight. The sediments were
then transported in insulated coolers. Upon arrival in the
laboratory, the sediments were still warm; they were homogenized and stored in airtight bottles under a N2:CO2 (80 : 20,
v/v) headspace at 90∞C.
bottles under N2. The bottles were sealed with butyl rubber
stoppers (Bellco Glass) and aluminum crimps. The bottles
were flushed with N2:CO2 (80:20, v/v) for 10 min and 3 ml of
artificial seawater was added. Killed controls were generated
by autoclaving (121∞C, 20 min) sediment slurries for three
consecutive days. When necessary, sodium molybdate was
added from concentrated anoxic stock solutions to obtain a
final concentration of 25 mM.
[14C] radiolabel studies
In separate radiotracer experiments, 0.05 mCi of [U-14C]-acetate (44.5 mCi mmol-1; 0.37 mM acetate), 0.045 mCi [ring14
C]-benzoate (8 mCi mmol-1; 1.9 mM benzoate), 0.05 mCi [U14
C]-glucose (50 mCi mmol-1; 0.33 mM glucose), or 0.05 mCi
L-[U-14C]-glutamate (180 mCi mmol-1; 92.5 nM L-glutamate)
was injected with a syringe and needle from 2.5 mCi ml-1
stock solutions in sterile anoxic water. Approximately
0.04 mCi of [U-14C]-palmitate (821 mCi mmol-1; 16.2 nM
palmitate) was added to small (7.5 mm diameter) disks of airdried sediment from a stock solution (821 mCi mmol-1 in toluene) using a Hamilton syringe. The disk was allowed to airdry momentarily and then placed inside the serum bottle
containing the sediment slurry. The serum bottle was then
flushed with N2:CO2 (80:20) for 10 min. The production of
14
CO2 over time was monitored by withdrawing headspace
samples (0.2–1.0 ml) and analysing them for 14CO2 as
described below.
In order to study the production of potential extracellular
intermediates isotope trapping studies were conducted as
follows. Sediment in serum bottles was amended with
0.015 ml of anoxic stock solutions containing potential intermediates to increase their concentrations as follows: acetate,
250 mM; propionate, 250 mM; butyrate, 250 mM; and lactate,
250 mM. Production of 14CO2 was monitored over time. In
order to determine if radiolabel accumulated in the potential
intermediates, the sediment samples were acidified with 1 ml
of 5 N sulphuric acid to stop metabolism and to convert
HCO3– to CO2. The headspace was flushed with N2 for 20 min
with mixing for 30 s with a vortex mixer every 5 min during
the flushing. Once the 14CO2 had been flushed out, pore
water was collected from a subsample (1.5 ml) of the sediment by centrifugation. The pore water was filtered (0.2 mm
pore diameter, PVDF; Alltech), and the levels of radioactivity
in the potential organic intermediates were determined by
radiochromatography as described below.
Analytical techniques
Sediment incubations
Artificial seawater contained the following constituents (in
grams per litre of deionised water): NaCl, 25.0; MgCl2·6H2O,
6.592; MgSO4·7H2O, 5.093; (NH4)2SO4, 1.000; NaHCO3,
0.145; CaCl2·2H2O, 1.500; KCl, 0.532; KH2PO4, 0.420; NaBr,
0.087; SrCl2·6H2O, 0.024; and H3BO3, 0.026. The artificial
seawater was degassed with N2:CO2 (80:20, v/v) and autoclaved (121∞C, 20 min). All gases used in the studies were
passed through a heated column of reduced copper fillings
to remove traces of oxygen. In an anaerobic chamber, sediments (10 g) were transferred into 30-ml Wheaton serum
Sulphate, sulphite, thiosulphate and nitrate concentrations
were determined by ion chromatography as previously
described (Lovley and Phillips, 1994). Elemental sulphur (S0)
was determined via reversed-phase HPLC after chloroform
extraction of sediments (Henshaw et al., 1997). Iron concentrations were determined as previously described (Lovley and
Phillips, 1986). The concentration of H2 was monitored over
time by headspace analysis with a reduction gas detector
(RGD2, Trace Analytical, Menlo Park, CA). Methane concentrations were determined via GC (Hewlett Packard, HP 6890
Series GC).
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 583–591
590 J. M. Tor, J. P. Amend and D. R. Lovley
To measure the production of 14CO2, headspace from the
incubation vessel was injected into a sealed scintillation vial
containing 1 ml of 0.5 N NaOH. The vial was gently stirred
every 5 min for 20 min. Ecolume (10 ml; ICN Biomedical,
Costa Mesa, CA) was added to the vials, and 14C was quantified by liquid scintillation counting. In addition, the production of 14CO2 and 14CH4 was monitored by analysing the
headspace via gas proportional counting, as previously
described (Lovley et al., 1995).
To measure the accumulation of short-chain fatty acids,
samples were extracted with a vacuum distillation procedure,
as previously described (Lovley and Klug, 1983). The potential short-chain fatty acids were separated by highperformance liquid chromatography (HPLC). As previously
described (Tor and Lovley, 2001), the filtrate was loaded onto
a Fast Acid Analysis column (100 ¥ 7.8 mm; Bio-Rad Laboratories) with an eluent of 5 mM sulphuric acid at a flow rate
of 0.7 ml min-1. The compounds were detected with a variable-wavelength UV detector (Shimadzu SPD-6 A) set at
210 nm. In metabolic intermediate studies, the fractions were
collected and counted by liquid scintillation counting.
Acknowledgements
The National Science Foundation LExEn Program supported
this research, grant MCB-0085365. We thank Sergio Gurrieri,
Salvo Inguaggiato, Toti Francofonte, Everett Shock and Karyn
Rogers for fruitful discussions and help with fieldwork on
Vulcano. We are also indebted to Rocco Favara and the staff
at the Istituto Nationale de Geofisica e Vulcanologia in Palermo, without whose assistance and friendship this work
would not have been possible.
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