71
wt), per unit volume (e.g. L), or on an areal basis (e.g. m
2
). For further details of
sample assay formats, analytical separation by chromatography and examples see
Capone (1993). Details about calculation of the Bunsen coefficient, which varies by
temperature and salinity, can be found in Breitbarth et al. (2004).
As mentioned in Chap. 2, the reduction of N 2 by conventional nitrogenase
requires 8e
−
and yields as products 2 molecules of NH 3 and one of H 2 . C 2 H 2 reduction in contrast requires only 2e
−
and does not yield H 2 as a major end-product.
Acetylene reduction rates are typically calibrated to N 2 fixation by a factor of 4
(assuming that no reductant goes to H 2 when C 2 H 2 is the substrate). However, it is
strongly recommended that C 2 H 2 reduction results be calibrated with
15
N 2 uptake
(see Sect. 5.4 below) if quantitative results are the objective.
The discovery of N 2 -fixing microorganisms that were small, relatively dilute and
difficult to visualize or concentrate resulted in less reliance on C 2 H 2 reduction and
more on stable isotopic approaches (see below) in oligotrophic waters because of
sensitivity and length of assay issues (Wilson et al. 2012).
In recent years, the C 2 H 2 reduction method has been modified and applied in a
variety of new ways. Sensitivity of analysis for C 2 H 4 detection has been improved
by using a Reduced Gas Analyzer (Wilson et al. 2012) as well as by use of highly
purified C 2 H 2 to minimize the C 2 H 4 background (Kitajima et al. 2009). A C 2 H 2 perfusion method was developed by Capone and Carpenter (1982) for assaying small
marine sediment cores. An on-line system for assaying cyanobacterial cultures by
flame ionization detector gas chromatography (FID-GC) by sampling the culture
gas phase with a rapidly cycling automated sample valve was reported by Staal et al.
(2001). They further improved on the sensitivity of the method by replacing the
traditional FID-GC with a laser-based gas detector which could be employed in a
continuous flow mode and provided three orders of magnitude better sensitivity.
However, obtaining high purity C 2 H 2 with low levels of C 2 H 4 contamination has
been a challenge. Most recently, a system for online continuous underway measurements on ships at sea was successfully developed and deployed using Cavity Ring
Down Laser Absorption Spectroscopy (CRDLAS) (Cassar et al. 2012, 2018).
In contrast to the conventional Mo nitrogenase, V nitrogenase reduces acetylene
to ethane rather than ethylene (Dilworth et al. 1987). A modified method using
13
C-labeled acetylene called the isotopic acetylene reduction assay (ISARA) makes
it possible to sensitively differentiate between conventional Mo nitrogenase activity
and alternative nitrogenases (Zhang et al. 2016).
5.4 Isotopic Tracers:
15
N
The use of enriched isotopic methods to document pathways of N metabolism was
first developed in the early 1940s by researchers at the University of Wisconsin
(Burris and Miller 1941; Wilson and Burris 1947) and was initially used in agricultural systems. Burris later went on to apply his methods in coral reef systems as well
(Burris 1976, 1983) (Table 5.1).
5.4 Isotopic Tracers:
15 N
wt), per unit volume (e.g. L), or on an areal basis (e.g. m
2
). For further details of
sample assay formats, analytical separation by chromatography and examples see
Capone (1993). Details about calculation of the Bunsen coefficient, which varies by
temperature and salinity, can be found in Breitbarth et al. (2004).
As mentioned in Chap. 2, the reduction of N 2 by conventional nitrogenase
requires 8e
−
and yields as products 2 molecules of NH 3 and one of H 2 . C 2 H 2 reduction in contrast requires only 2e
−
and does not yield H 2 as a major end-product.
Acetylene reduction rates are typically calibrated to N 2 fixation by a factor of 4
(assuming that no reductant goes to H 2 when C 2 H 2 is the substrate). However, it is
strongly recommended that C 2 H 2 reduction results be calibrated with
15
N 2 uptake
(see Sect. 5.4 below) if quantitative results are the objective.
The discovery of N 2 -fixing microorganisms that were small, relatively dilute and
difficult to visualize or concentrate resulted in less reliance on C 2 H 2 reduction and
more on stable isotopic approaches (see below) in oligotrophic waters because of
sensitivity and length of assay issues (Wilson et al. 2012).
In recent years, the C 2 H 2 reduction method has been modified and applied in a
variety of new ways. Sensitivity of analysis for C 2 H 4 detection has been improved
by using a Reduced Gas Analyzer (Wilson et al. 2012) as well as by use of highly
purified C 2 H 2 to minimize the C 2 H 4 background (Kitajima et al. 2009). A C 2 H 2 perfusion method was developed by Capone and Carpenter (1982) for assaying small
marine sediment cores. An on-line system for assaying cyanobacterial cultures by
flame ionization detector gas chromatography (FID-GC) by sampling the culture
gas phase with a rapidly cycling automated sample valve was reported by Staal et al.
(2001). They further improved on the sensitivity of the method by replacing the
traditional FID-GC with a laser-based gas detector which could be employed in a
continuous flow mode and provided three orders of magnitude better sensitivity.
However, obtaining high purity C 2 H 2 with low levels of C 2 H 4 contamination has
been a challenge. Most recently, a system for online continuous underway measurements on ships at sea was successfully developed and deployed using Cavity Ring
Down Laser Absorption Spectroscopy (CRDLAS) (Cassar et al. 2012, 2018).
In contrast to the conventional Mo nitrogenase, V nitrogenase reduces acetylene
to ethane rather than ethylene (Dilworth et al. 1987). A modified method using
13
C-labeled acetylene called the isotopic acetylene reduction assay (ISARA) makes
it possible to sensitively differentiate between conventional Mo nitrogenase activity
and alternative nitrogenases (Zhang et al. 2016).
5.4 Isotopic Tracers:
15
N
The use of enriched isotopic methods to document pathways of N metabolism was
first developed in the early 1940s by researchers at the University of Wisconsin
(Burris and Miller 1941; Wilson and Burris 1947) and was initially used in agricultural systems. Burris later went on to apply his methods in coral reef systems as well
(Burris 1976, 1983) (Table 5.1).
5.4 Isotopic Tracers:
15 N