Environmental Engineering Reference
In-Depth Information
anammox [11]: 1)
15
N-labeled N
2
accumulated linearly without a lag phase
in incubations with
15
NH
4
+
, indicating that the label was not transformed via
intermediates such as NO
2
−
or NO
3
−
;2)
15
N from NH
4
+
was recovered as
14
N
15
N rather than
15
N
15
N, consistent with isotope pairing during anammox
[75] and inconsistent with a direct oxidation coupled to Mn reduction; and 3)
the production of
15
N-labeled N
2
from
15
NO
3
−
was stimulated by the addition
of NH
4
+
. Furthermore, it occurred with a lag phase consistent with a gradual
labeling of the NO
2
−
-pool, with NO
2
−
being the direct oxidant for NH
4
+
. These
three characteristics, possibly supplemented with results from incubations with
15
NO
2
−
[12], form a checklist of criteria for the demonstration of anammox
with
15
N-techniques.
N-15-labelling provides a very sensitive technique for the determination of
anammox rates. For example, rates of the order of 1 nM h
−
1
were determined in
incubations over
2 days with water from Golfo Dulce [11]. As an additional
advantage, incubations with
15
NO
3
−
or
15
NO
2
−
allow for the simultaneous
determination of anammox and denitrification, giving insights to the relative
importance of the two sinks for fixed N [70]. This partitioning rests on the char-
acteristic patterns of isotope pairing in N
2
formed through the two pathways.
In contrast to the 1-to-1 pairing of nitrogen atoms from nitrite and ammonium
during anammox, the reduction of two equivalent molecules of nitrate or nitrite
via nitric oxide to nitrous oxide results in random pairing of the isotopes in N
2
formed through denitrification, with
14
N
14
N,
14
N
15
N, and
15
N
15
N forming at a
ratio of (1 - F)
2
:2F(1 - F):(1 - F)
2
, where F is the fraction of
15
N in the nitrogen
source [29, 41, 84]. Thus, in experiments with
15
NO
3
−
or
15
NO
2
−
and
14
NH
4
+
,
15
N
15
N only forms through denitrification, and total denitrification as well as
the production of
14
N
15
N through this pathway can be calculated from the ratio
above. Subsequently the production of
14
N
15
N through anammox is quantified
as the difference between measured
14
N
15
N production and the production of
this mass through denitrification, and
14
N
14
N production though anammox is
calculated from this excess and an expected RATIO of
∼
14
N
14
N:
14
N
15
N from
anammox of (1 - F):F [70].
The accuracy of the
15
NO
3
−
/
15
NO
2
−
incubation approach depends critically
on an accurate determination of the fraction of
15
N (F) in the electron acceptor,
nitrate or nitrite, utilized by the two processes. Kinetic isotope effects have
been neglected in the calculations as they should bias the results by a few
percent at most. The available data suggests that the increase in NO
3
−
or
NO
2
−
concentrations through the amendments does not influence the measured
rates. Thus, apparent half-saturation concentrations (K
m
) with respect to NO
2
−
for both anammox and denitrification are low (
<
3 µM) [12, 73] compared
to the natural concentrations of these species, and anammox appears to be
NH
4
+
-limited in anoxic waters [11]. Although rates determined in incubations
with
15
NH
4
+
may thus overestimate the rates
in situ
, they are valuable as
