69
5.3 The Acetylene Reduction Method
The acetylene (C 2 H 2 ) reduction method was introduced in 1967 as a simple and
convenient method to detect and quantify nitrogenase activity in the environment
(Hardy et al. 1968; Stewart et al. 1967) (Table 5.1). Acetylene is a substrate analog
of dinitrogen gas, and the conventional (Mo containing) nitrogenase enzyme reduces
it to ethylene (C 2 H 4 ) (Seefeldt et al. 2020), which is easily separated and quantified
by gas chromatography (Capone 1993). The introduction of this method led to a
rapid expansion in studies of N 2 fixation in a range of marine ecosystems including
the open ocean, coral reefs, benthic seagrasses and macroalgae (see Carpenter and
Capone 2008). In particular, Carpenter and his colleagues undertook a series of
cruises in the early 1970s to the Sargasso (SW North Atlantic) and Caribbean Seas
(Carpenter 1973; Carpenter and McCarthy 1975; Carpenter and Price 1977) detailing the distribution and importance of Trichodesmium in these systems.
Acetylene is reduced to ethylene by the nitrogenase enzyme but reduces less H
+
to H 2 than when N 2 is the substrate. It is a noncompetitive inhibitor of nitrogenase
(Rivera-Ortiz and Burris 1975; Seefeldt et al. 2020), which means that N 2 is not
fixed when C 2 H 2 is used to measure N 2 fixation, although the reaction is dependent
on relative substrate concentrations. Hence, C 2 H 2 “starves” N 2 fixers for nutritional
N. Moreover, C 2 H 2 actively inhibits a broad range of physiological types including
ammonia and methane oxidizing bacteria, methylotrophs and methanogens (see
Oremland and Capone 1988).
Briefly, C 2 H 2 is typically added to the gas phase (final volume 10–20%) of a
culture or natural sample being assayed in a sealed vessel with a sampling port for
withdrawing gas samples (Fig. 5.2). Acetylene is highly soluble in the aqueous
phase and readily dissolves after its addition. With time, samples of the gas phase of
the assay are subsampled with a gas tight syringe and analyzed by solid phase gas
chromatography for the appearance of C 2 H 4 which is quantified by comparison with
Fig. 5.1 Cultivated isolates of marine diazotrophs. (a) Cyanobacteria (b) Microaerophilic heterotrophs in agarose stabs. (Courtesy L. Riemann)
5.3 The Acetylene Reduction Method
5.3 The Acetylene Reduction Method
The acetylene (C 2 H 2 ) reduction method was introduced in 1967 as a simple and
convenient method to detect and quantify nitrogenase activity in the environment
(Hardy et al. 1968; Stewart et al. 1967) (Table 5.1). Acetylene is a substrate analog
of dinitrogen gas, and the conventional (Mo containing) nitrogenase enzyme reduces
it to ethylene (C 2 H 4 ) (Seefeldt et al. 2020), which is easily separated and quantified
by gas chromatography (Capone 1993). The introduction of this method led to a
rapid expansion in studies of N 2 fixation in a range of marine ecosystems including
the open ocean, coral reefs, benthic seagrasses and macroalgae (see Carpenter and
Capone 2008). In particular, Carpenter and his colleagues undertook a series of
cruises in the early 1970s to the Sargasso (SW North Atlantic) and Caribbean Seas
(Carpenter 1973; Carpenter and McCarthy 1975; Carpenter and Price 1977) detailing the distribution and importance of Trichodesmium in these systems.
Acetylene is reduced to ethylene by the nitrogenase enzyme but reduces less H
+
to H 2 than when N 2 is the substrate. It is a noncompetitive inhibitor of nitrogenase
(Rivera-Ortiz and Burris 1975; Seefeldt et al. 2020), which means that N 2 is not
fixed when C 2 H 2 is used to measure N 2 fixation, although the reaction is dependent
on relative substrate concentrations. Hence, C 2 H 2 “starves” N 2 fixers for nutritional
N. Moreover, C 2 H 2 actively inhibits a broad range of physiological types including
ammonia and methane oxidizing bacteria, methylotrophs and methanogens (see
Oremland and Capone 1988).
Briefly, C 2 H 2 is typically added to the gas phase (final volume 10–20%) of a
culture or natural sample being assayed in a sealed vessel with a sampling port for
withdrawing gas samples (Fig. 5.2). Acetylene is highly soluble in the aqueous
phase and readily dissolves after its addition. With time, samples of the gas phase of
the assay are subsampled with a gas tight syringe and analyzed by solid phase gas
chromatography for the appearance of C 2 H 4 which is quantified by comparison with
Fig. 5.1 Cultivated isolates of marine diazotrophs. (a) Cyanobacteria (b) Microaerophilic heterotrophs in agarose stabs. (Courtesy L. Riemann)
5.3 The Acetylene Reduction Method