or to a series of complex metabolic reactions comprising
several steps where polymers are hydrolyzed into simple
soluble compounds (dissolved organic nitrogen currently
known as DON) such as amines, amino acids, urea, and
pyrimidines (see Sect. 14.2).
In the absence of specific method to quantify the process
of ammonification, most authors quantify this process as
follows:
1. In situ via incubation of natural samples and
measurements of the kinetic of ammonium production;
2. In laboratory experiments via measurements of the kinetics of ammonium production or using techniques of isotopic dilution (Box 14.10).
Box 14.10: Methods for Determining the Denitrifying
Activity: Approaches for a Difficult Problem
Patricia Bonin
Denitrification, the reduction of nitrate to nitrite
and gaseous products (NO, N 2 O, and N 2 ) is an important microbial process regulating both primary
production through the availability of nitrates, the
water quality and chemistry of the atmosphere, both
locally and globally. Unfortunately, quantifying this
process on a natural sample from the soil or aquatic
environments is very complex. This is mainly due to the
fact that the substrate denitrification can be used in
many biological processes and the final product (N 2 )
is the major component of the atmosphere.
The main approaches developed to quantify
denitrification are presented and discussed in this
part.
Methods involving the quantification of the consumption of nitrate (substrate for denitrification) are
unreliable because depending of the ecosystems
denitrifying activity may be either (1) overestimated,
nitrates are used by processes other than denitrification
such as dissimilatory reduction to ammonium or (2)
underestimated since the production of nitrate through
the nitrification-denitrification coupling is not taken
into account or assimilation The calculations of nitrate
fluxes from nitrate profiles determined in the pore
water lead to the same problem. Moreover, in most
cases only the passive diffusion of nitrate is taken into
account and not the diffusion resulting from sediment
reworking.
(continued)
Box 14.10 (continued)
Other methods are based on the quantification of
the products of denitrification, N 2 O or N 2 , have been
developed. Direct measurement of di-nitrogen production is difficult because nitrogen (molar mass 14 g/
mol) is the main component of air (78.09 %). In some
studies, the authors incubated their samples under
atmosphere without N 2 , by bubbling vials with an
inert gas such as argon or helium, to directly quantify
the production of N 2 . But this operation is difficult
because N 2 contaminations are difficult to control.
Other authors quantify the evolution of N 2 /Ar ratio
mass spectrometry. Another type of approach involves
the use of a stable isotope of nitrogen, the heavy
nitrogen (molar mass of 15 g/mol). This isotope,
accounting for only 0.36 % of the total nitrogen is in
low quantities in the natural environment. Different
isotopic methods have been developed. These labelling methods are more accurate and sensitive than
previous methods, but are more expensive and difficult
to implement. Different approaches based on the use
of
15 N were used: methods of isotopic fractionation,
isotope dilution or direct measurement of denitrification by mass spectrometry assay of 28/29/30 N 2
formed from
15 NH 4 or
15 NO 3 added.
Ace ´ tyle ` ne Inhibition Technique (AIT)
The development of inhibition method developed for
the study of terrestrial and aquatic ecosystems has
stimulated research on nitrogen cycle in the late
1970s. Acetylene amendment allows inhibition of the
last step of denitrification, that is the reduction of
nitrous oxide by di-nitrogen, and allows assimilating
to the speed of accumulation of nitrous oxide to
denitrifying activity (Yoshinari and Knowles 1976).
Quantification of nitrous oxide concentration in the
samples is facilitated by gas chromatography (electron
capture detector). This method is inexpensive, easy to
implement and not too time consuming (Box
Fig. 14.16).
However, the limitations of this method, did not
take long to be demonstrated: (1) incomplete inhibition of nitrous oxide reduction in the presence of low
nitrate concentrations and/or high organic matter
(continued)
570
J.-C. Bertrand et al.
several steps where polymers are hydrolyzed into simple
soluble compounds (dissolved organic nitrogen currently
known as DON) such as amines, amino acids, urea, and
pyrimidines (see Sect. 14.2).
In the absence of specific method to quantify the process
of ammonification, most authors quantify this process as
follows:
1. In situ via incubation of natural samples and
measurements of the kinetic of ammonium production;
2. In laboratory experiments via measurements of the kinetics of ammonium production or using techniques of isotopic dilution (Box 14.10).
Box 14.10: Methods for Determining the Denitrifying
Activity: Approaches for a Difficult Problem
Patricia Bonin
Denitrification, the reduction of nitrate to nitrite
and gaseous products (NO, N 2 O, and N 2 ) is an important microbial process regulating both primary
production through the availability of nitrates, the
water quality and chemistry of the atmosphere, both
locally and globally. Unfortunately, quantifying this
process on a natural sample from the soil or aquatic
environments is very complex. This is mainly due to the
fact that the substrate denitrification can be used in
many biological processes and the final product (N 2 )
is the major component of the atmosphere.
The main approaches developed to quantify
denitrification are presented and discussed in this
part.
Methods involving the quantification of the consumption of nitrate (substrate for denitrification) are
unreliable because depending of the ecosystems
denitrifying activity may be either (1) overestimated,
nitrates are used by processes other than denitrification
such as dissimilatory reduction to ammonium or (2)
underestimated since the production of nitrate through
the nitrification-denitrification coupling is not taken
into account or assimilation The calculations of nitrate
fluxes from nitrate profiles determined in the pore
water lead to the same problem. Moreover, in most
cases only the passive diffusion of nitrate is taken into
account and not the diffusion resulting from sediment
reworking.
(continued)
Box 14.10 (continued)
Other methods are based on the quantification of
the products of denitrification, N 2 O or N 2 , have been
developed. Direct measurement of di-nitrogen production is difficult because nitrogen (molar mass 14 g/
mol) is the main component of air (78.09 %). In some
studies, the authors incubated their samples under
atmosphere without N 2 , by bubbling vials with an
inert gas such as argon or helium, to directly quantify
the production of N 2 . But this operation is difficult
because N 2 contaminations are difficult to control.
Other authors quantify the evolution of N 2 /Ar ratio
mass spectrometry. Another type of approach involves
the use of a stable isotope of nitrogen, the heavy
nitrogen (molar mass of 15 g/mol). This isotope,
accounting for only 0.36 % of the total nitrogen is in
low quantities in the natural environment. Different
isotopic methods have been developed. These labelling methods are more accurate and sensitive than
previous methods, but are more expensive and difficult
to implement. Different approaches based on the use
of
15 N were used: methods of isotopic fractionation,
isotope dilution or direct measurement of denitrification by mass spectrometry assay of 28/29/30 N 2
formed from
15 NH 4 or
15 NO 3 added.
Ace ´ tyle ` ne Inhibition Technique (AIT)
The development of inhibition method developed for
the study of terrestrial and aquatic ecosystems has
stimulated research on nitrogen cycle in the late
1970s. Acetylene amendment allows inhibition of the
last step of denitrification, that is the reduction of
nitrous oxide by di-nitrogen, and allows assimilating
to the speed of accumulation of nitrous oxide to
denitrifying activity (Yoshinari and Knowles 1976).
Quantification of nitrous oxide concentration in the
samples is facilitated by gas chromatography (electron
capture detector). This method is inexpensive, easy to
implement and not too time consuming (Box
Fig. 14.16).
However, the limitations of this method, did not
take long to be demonstrated: (1) incomplete inhibition of nitrous oxide reduction in the presence of low
nitrate concentrations and/or high organic matter
(continued)
570
J.-C. Bertrand et al.