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Exercise 7
Organic Nitrogen
A large fraction of the total nitrogen in fresh waters may occur as organic nitrogen in
particulate, and especially in dissolved, form. While these organic nitrogen compounds
occur largely in particulate and dissolved organic detritus and generally are not
available to photosynthetic organisms, they represent a major reservoir of nitrogen in
aquatic ecosystems.
A number of analytical techniques are available for the determination of particulate
and dissolved organic nitrogen. However, many of the modern methods employ
elaborate instrumentation (see the "Automated Analyses" section below).
Particulate and dissolved organic nitrogen are usually separated by filtration in allglass apparatus with glass fiber filters (Reeve Angel 984H, 0.5-llm pore size or
Whatman GF/F, 0.6 to 0.7-llm pore size). The fractions are then analyzed by different
techniques.
The nitrogen in the particulate matter is converted to ammonia by the classical
Kjeldahl method, after treatment with sulfuric acid. The resulting ammonia is then
concentrated in a closed system by distillation and titrated or analyzed absorptiometrically by complex formation with ninhydrin without prior distillation (Strickland and
Parsons, 1968). Alternatively, the particulate matter is combusted at high temperature
in a C-H-N analyzer; in this case the organic nitrogen is converted to free N2 and then
analyzed by gas chromatography.
Dissolved organic nitrogen concentrations are more difficult to determine in water.
The micro-Kjeldahl technique has been employed [e.g., Stadelmann (1971)], but
sensitivity limits often exceed the low concentrations found in many natural waters.
More recently organic nitrogen compounds and ammonia have been analyzed by
photolytic decomposition and oxidation with high intensity, ultra violet (UV) radiation
(Armstrong and Tibbitts, 1968; Manny et al., 1971; Stainton et al., 1977). Duration of
combustion, pH, and adequate oxygen affect the photolytic oxidation and must be
controlled carefully. The products of photooxidation are then measured as ammonium,
nitrate, and nitrite. When the concentrations of the inorganic nitrogen compounds in
the samples prior to UV irradiation are known, an estimate of dissolved organic
nitrogen can be made. This method is especially useful when the samples contain low
levels of organic nitrogen. (See also the "Automated Analyses" section below for
additional procedures for nitrogen species.)
NITROGEN FIXATION
Investigations during the past two decades have demonstrated that fixation of
molecular nitrogen (N 2 ) by microflora can serve as a major input of nitrogen to aquatic
ecosystems under certain conditions [cf., review in Wetzel (1983)]. Nitrogen fixation is
primarily light-dependent in that it requires hydrogen acceptors and adenosine
triphosphate (A TP), both of which are generated in photosynthesis. Nitrogen fixation
in aquatic systems is restricted almost exclusively to heterocystous blue-green algae
[but see Gallon et al. (1975)] and photosynthetic bacteria.
Several methods have been employed to assay rates of N2 fixation [reviewed in
Hardy et al. (1973)]. While the use of the stable isotope, 15N2, in enrichment
experiments is preferred, 15N2 is expensive, and detection by mass or optical emission
spectrometry requires elaborate instrumentation. Another approach is to provide an
alternate substrate, such as acetylene, for the active enzyme, nitrogenase. The assay is
based on nitrogenase-catalyzed reduction of acetylene (C 2 H 2 ) to ethylene (C z H4), gas
Exercise 7
Organic Nitrogen
A large fraction of the total nitrogen in fresh waters may occur as organic nitrogen in
particulate, and especially in dissolved, form. While these organic nitrogen compounds
occur largely in particulate and dissolved organic detritus and generally are not
available to photosynthetic organisms, they represent a major reservoir of nitrogen in
aquatic ecosystems.
A number of analytical techniques are available for the determination of particulate
and dissolved organic nitrogen. However, many of the modern methods employ
elaborate instrumentation (see the "Automated Analyses" section below).
Particulate and dissolved organic nitrogen are usually separated by filtration in allglass apparatus with glass fiber filters (Reeve Angel 984H, 0.5-llm pore size or
Whatman GF/F, 0.6 to 0.7-llm pore size). The fractions are then analyzed by different
techniques.
The nitrogen in the particulate matter is converted to ammonia by the classical
Kjeldahl method, after treatment with sulfuric acid. The resulting ammonia is then
concentrated in a closed system by distillation and titrated or analyzed absorptiometrically by complex formation with ninhydrin without prior distillation (Strickland and
Parsons, 1968). Alternatively, the particulate matter is combusted at high temperature
in a C-H-N analyzer; in this case the organic nitrogen is converted to free N2 and then
analyzed by gas chromatography.
Dissolved organic nitrogen concentrations are more difficult to determine in water.
The micro-Kjeldahl technique has been employed [e.g., Stadelmann (1971)], but
sensitivity limits often exceed the low concentrations found in many natural waters.
More recently organic nitrogen compounds and ammonia have been analyzed by
photolytic decomposition and oxidation with high intensity, ultra violet (UV) radiation
(Armstrong and Tibbitts, 1968; Manny et al., 1971; Stainton et al., 1977). Duration of
combustion, pH, and adequate oxygen affect the photolytic oxidation and must be
controlled carefully. The products of photooxidation are then measured as ammonium,
nitrate, and nitrite. When the concentrations of the inorganic nitrogen compounds in
the samples prior to UV irradiation are known, an estimate of dissolved organic
nitrogen can be made. This method is especially useful when the samples contain low
levels of organic nitrogen. (See also the "Automated Analyses" section below for
additional procedures for nitrogen species.)
NITROGEN FIXATION
Investigations during the past two decades have demonstrated that fixation of
molecular nitrogen (N 2 ) by microflora can serve as a major input of nitrogen to aquatic
ecosystems under certain conditions [cf., review in Wetzel (1983)]. Nitrogen fixation is
primarily light-dependent in that it requires hydrogen acceptors and adenosine
triphosphate (A TP), both of which are generated in photosynthesis. Nitrogen fixation
in aquatic systems is restricted almost exclusively to heterocystous blue-green algae
[but see Gallon et al. (1975)] and photosynthetic bacteria.
Several methods have been employed to assay rates of N2 fixation [reviewed in
Hardy et al. (1973)]. While the use of the stable isotope, 15N2, in enrichment
experiments is preferred, 15N2 is expensive, and detection by mass or optical emission
spectrometry requires elaborate instrumentation. Another approach is to provide an
alternate substrate, such as acetylene, for the active enzyme, nitrogenase. The assay is
based on nitrogenase-catalyzed reduction of acetylene (C 2 H 2 ) to ethylene (C z H4), gas