65
Table 5.1 Methods to study N
2 -fixers and N
2 fixation
Target
Instrumentation & needs
Notes
References
1. Growth/physiology
A. Growth on N-free media
Diazotroph growth
and isolation
N-free media
Difficulties in growing
uncultivated microbes with
unknown needs
Traditional/historic
B. N increase, NH
3 production
Diazotroph growth Nutrient analysis
Imprecise, prone to potential
artifact, e.g. reagent N
contamination
Traditional/historic
C. Tracer isotope uptake
13
N
2 uptake,
(
13
N
2 - > 
13
NH
3 - > amino
acids etc.)
N
2 fixation rate
Cyclotron or linear accelerator Access & availability issues
Meeks (1978) and Wolk
et al. (1974)
15
N
2 uptake
(
15
N
2 - > 
15
NH
3 - > amino
acids or particulate matter)
N
2 fixation rate
IR Mass Spec.
Direct assay of N
2 fixation
Dugdale et al. (1961) and
Montoya et al. (1996)
D. Analog assay
C
2 H
2 reduction
(HC ≡ CH - > H
2 C=CH
2 )
Nitrogenase
activity/rate
Gas chromatograph or reduced
gas analyzer
Indirect, requires conversion
simple enzymatic assay
Capone (1993), Foster
et al. (2011), Hardy et al.
(1968) and Stewart et al.
(1967)
Isotopic C
2 H
2 reduction
(H
13
C ≡ 
13
CH - > H
2
13
C =
13
CH
2 )
Nitrogenase
activity
IR Mass Spec.
Can differentiate amongst
nitrogenases
Dekas et al. (2016),
Dupouy et al. (2000),
Foster et al. (2011) and
Zhang et al. (2016)
E. H
2
H
2 measurements
H
2 evolved by
nitrogenase
Gas chromatograph or reduced
gas analyzer
Easy to measure, but multiple
sources and sinks
Moore et al. (2009)
(continued)
5.2 Cultivation