5
Bacteria and Marine Biogeochemistry
198
and
15
N
15
N, it has been possible to calculate not
only the rate of denitrification, but even to
discriminate between denitrification from nitrate
diffusing down from the overlying seawater and
denitrification from nitrate formed by nitrification
within the sediment. The results have shown that
at the normal, low concentrations of nitrate in
seawater, <10-20 µM, the main source of nitrate for
denitrification is the internally formed nitrate
derived from nitrification.
5.6.3
Example: Sulfate Reduction
As an example of a radio-tracer method, the
measurement of sulfate reduction rates using
35
SO 4
2–
according to Jørgensen (1978); Fossing
and Jørgensen (1989); Kallmeyer et al. (2004) is
described in brief (Fig. 5.14). The
35
SO 4
2–
is
injected with a microsyringe (1) into whole,
intact sediment cores in quantities of a few
microliters which contain ca 100 kBq
(1 becquerel = 1 radioactive disintegration per
second; 37 kBq = 1 microcurie). After 4-8 hours
the core is sectioned (2), and the sediment
samples are fixed with zinc acetate. The zinc
binds the sulfide and prevents its oxidation and
it also kills the bacteria and prevents further
sulfate reduction. The reduced
35
S is then
separated from the sediment by acidification (3),
and the released H 2
35
S is transferred in a stream
of N 2 to a trap containing zinc acetate where
Zn
35
S precipitates. The radioactivities of the
Zn
35
S and of the remaining
35
SO 4
2–
are then
analyzed by liquid scintillation counting (4). In
a sediment core the concentration gradient of
sulfate in the pore water is analyzed (5), and the
rates of sulfate reduction can then be calculated
according to the equation:
Sulfate reduction rate =
( )
t
24
SO
S
H
SO
-
2
4
35
35
2
-
2
4
⋅
⋅
φ
·1.06 nmol SO 4
2- cm -3 day -1
(5.29)
where φ is porosity, (SO 4
2–
) is sulfate concentration in the pore water ( φ (SO 4
2–
) is then sulfate
concentration per volume sediment, H 2
35
S is
radioactivity of total reduced sulfur),
35
SO 4
2–
is
radioactivity of added sulfate tracer, t is experiment time in hours, and 1.06 is a correction factor
for the small dynamic isotope discrimination by
the bacteria against the heavier isotope. One nmol
(nanomol) is 10
-9
mol. This formula is a good
approximation as long as only a small fraction of
the labeled sulfate is reduced during incubation, a
condition normally fulfilled in marine sediments.
Sulfate reduction rates in marine shelf sediments commonly lie in the range of 1-100 nmol SO 4
2–
cm
–3
day
–1
(Jørgensen 1982). Since the sulfate concentration in the pore water is around 28 mM or
20 µmol cm
–3
, the turn-over time of the sulfate pool
is in the order of 1-50 years. A purely chemical
experiment would thus require a month to several
years of incubation. This clearly illustrates why a
radiotracer technique is required to measure the
rate within several hours. In sediment cores from
the open eastern equatorial Pacific obtained by
the Ocean Drilling Program it has been possible to
push the detectability of this radiotracer method
to its physical limit by measuring sulfate reduction
rates of <0.001 nmol cm
-3
d
-1
in 9 million year old
sediments at 300 m below the seafloor (Parkes et
al. 2005).
Similar principles as described here are used in
measurements of, e.g. the oxidation of
14
C-labelled
methane or the formation of methane from
14
Clabelled CO 2 or acetate. Often there are no
gaseous substrates or products, however, and it is
not possible to separate the radioisotopes as efficiently as by the measurement of sulfate reduction
or methane formation and oxidation. This is the
case for radiotracer studies of intermediates in the
mineralization of organic matter in sediments, e.g.
of
14
C-labelled sugars, amino acids, or volatile
fatty acids, studies which have been important for
the understanding of the pathways of organic
degradation (Fig. 5.11). These organic compounds, however, occur at low concentrations and
have a fast turn-over of minutes to hours, so the
sensitivity is less important. More important is the
fact that these compounds are at a steady-state
between production and consumption, which
means that their concentration may not change
during incubation in spite of their fast turnover. A
simple chemical experiment would thus not be able
to detect their natural dynamics, but a radiotracer
experiment may.
5.6.4
Specific Inhibitors
This limitation of chemical experiments is often
overcome by the use of a specific inhibitor (Oremland
and Capone 1988). The principle of an inhibitor technique may be, a) to block a sequence of processes at
a certain step in order to observe the accumulation of
an intermediate compound, b) to inhibit a certain
Bacteria and Marine Biogeochemistry
198
and
15
N
15
N, it has been possible to calculate not
only the rate of denitrification, but even to
discriminate between denitrification from nitrate
diffusing down from the overlying seawater and
denitrification from nitrate formed by nitrification
within the sediment. The results have shown that
at the normal, low concentrations of nitrate in
seawater, <10-20 µM, the main source of nitrate for
denitrification is the internally formed nitrate
derived from nitrification.
5.6.3
Example: Sulfate Reduction
As an example of a radio-tracer method, the
measurement of sulfate reduction rates using
35
SO 4
2–
according to Jørgensen (1978); Fossing
and Jørgensen (1989); Kallmeyer et al. (2004) is
described in brief (Fig. 5.14). The
35
SO 4
2–
is
injected with a microsyringe (1) into whole,
intact sediment cores in quantities of a few
microliters which contain ca 100 kBq
(1 becquerel = 1 radioactive disintegration per
second; 37 kBq = 1 microcurie). After 4-8 hours
the core is sectioned (2), and the sediment
samples are fixed with zinc acetate. The zinc
binds the sulfide and prevents its oxidation and
it also kills the bacteria and prevents further
sulfate reduction. The reduced
35
S is then
separated from the sediment by acidification (3),
and the released H 2
35
S is transferred in a stream
of N 2 to a trap containing zinc acetate where
Zn
35
S precipitates. The radioactivities of the
Zn
35
S and of the remaining
35
SO 4
2–
are then
analyzed by liquid scintillation counting (4). In
a sediment core the concentration gradient of
sulfate in the pore water is analyzed (5), and the
rates of sulfate reduction can then be calculated
according to the equation:
Sulfate reduction rate =
( )
t
24
SO
S
H
SO
-
2
4
35
35
2
-
2
4
⋅
⋅
φ
·1.06 nmol SO 4
2- cm -3 day -1
(5.29)
where φ is porosity, (SO 4
2–
) is sulfate concentration in the pore water ( φ (SO 4
2–
) is then sulfate
concentration per volume sediment, H 2
35
S is
radioactivity of total reduced sulfur),
35
SO 4
2–
is
radioactivity of added sulfate tracer, t is experiment time in hours, and 1.06 is a correction factor
for the small dynamic isotope discrimination by
the bacteria against the heavier isotope. One nmol
(nanomol) is 10
-9
mol. This formula is a good
approximation as long as only a small fraction of
the labeled sulfate is reduced during incubation, a
condition normally fulfilled in marine sediments.
Sulfate reduction rates in marine shelf sediments commonly lie in the range of 1-100 nmol SO 4
2–
cm
–3
day
–1
(Jørgensen 1982). Since the sulfate concentration in the pore water is around 28 mM or
20 µmol cm
–3
, the turn-over time of the sulfate pool
is in the order of 1-50 years. A purely chemical
experiment would thus require a month to several
years of incubation. This clearly illustrates why a
radiotracer technique is required to measure the
rate within several hours. In sediment cores from
the open eastern equatorial Pacific obtained by
the Ocean Drilling Program it has been possible to
push the detectability of this radiotracer method
to its physical limit by measuring sulfate reduction
rates of <0.001 nmol cm
-3
d
-1
in 9 million year old
sediments at 300 m below the seafloor (Parkes et
al. 2005).
Similar principles as described here are used in
measurements of, e.g. the oxidation of
14
C-labelled
methane or the formation of methane from
14
Clabelled CO 2 or acetate. Often there are no
gaseous substrates or products, however, and it is
not possible to separate the radioisotopes as efficiently as by the measurement of sulfate reduction
or methane formation and oxidation. This is the
case for radiotracer studies of intermediates in the
mineralization of organic matter in sediments, e.g.
of
14
C-labelled sugars, amino acids, or volatile
fatty acids, studies which have been important for
the understanding of the pathways of organic
degradation (Fig. 5.11). These organic compounds, however, occur at low concentrations and
have a fast turn-over of minutes to hours, so the
sensitivity is less important. More important is the
fact that these compounds are at a steady-state
between production and consumption, which
means that their concentration may not change
during incubation in spite of their fast turnover. A
simple chemical experiment would thus not be able
to detect their natural dynamics, but a radiotracer
experiment may.
5.6.4
Specific Inhibitors
This limitation of chemical experiments is often
overcome by the use of a specific inhibitor (Oremland
and Capone 1988). The principle of an inhibitor technique may be, a) to block a sequence of processes at
a certain step in order to observe the accumulation of
an intermediate compound, b) to inhibit a certain