175
The
15
N method coupled with nanoscale secondary ion mass spectrometry
(nanoSIMS) has also been used to measure cell-specific rates of N 2 fixation.
New methods continue to be developed. Since H 2 is evolved by nitrogenase, H 2
flux can be related to N 2 fixation activity. H 2 gas measurements are relatively simple
to make and can be used to obtain shipboard data in almost real time. A modified
method using
13
C-labeled acetylene called the isotopic acetylene reduction assay
(ISARA) makes it possible to measure N 2 fixation by alternative nitrogenases, as
well as that catalyzed by the Mo nitrogenase.
Since only a few of the diazotrophs can be easily identified visually, either by the
naked eye (e.g. Trichodesmium) or microscopically (e.g. Richelia), molecular biological methods have been used to identify and quantify diazotrophs in a broad
range of marine habitats. The introduction of the polymerase chain reaction (PCR)
led to a method using degenerate nitrogenase (usually nifH) primers to amplify nif
genes from uncultivated microorganisms, thereby enabling the discovery of new
organisms and establishing the true diversity of diazotrophs in the sea. More
recently, the advent of metagenomics and metatranscriptomics has provided unbiased approaches to cataloguing nif genes, although diazotrophs are generally in low
abundance. Sequence types representing specific lineages can be targeted with customized probes: using these specific probes, quantitative PCR (qPCR) has provided
much of the recent data on distributions, abundances and gene expression (RT-qPCR)
of uncultivated microorganisms including UCYN-A and NCDs.
Uncultivated diazotrophs have been visualized using fluorescent in situ hybridization (FISH) often coupled to catalyzed reporter deposition (CARD-FISH) to
enhance detection. This technique targets rRNA, thus the rRNA gene sequence must
be known, which it is not for many uncultivated diazotrophs that are only known by
their amplified nifH sequence. Also, CARD-FISH effectiveness is limited by cell
abundances: since diazotroph densities can be low it can be difficult to obtain sufficient numbers of cells on a filter to obtain good quantitative estimates.
Another approach for identifying diazotrophs termed Stable Isotope Probing
(SIP) is based on the increased density of genomic DNA labeled with
15
N. DNA is
extracted from samples incubated with enriched
15
N 2 gas, and the heavier labeled
DNA separated by ultracentrifugation. Sequencing of DNA from the heavier fractions is presumptively from diazotrophic organisms that have synthesized DNA (or
in some applications RNA) using the N from heavy labelled
15
N 2 gas. This method
is not quantitative, but does directly identify gene sequences associated with active
N 2 -fixing microorganisms, if enriched sufficiently. Several studies of marine N 2
fixation have coupled SIP with nanoSIMS which may allow for quantitative
interpretation.
The methods for directly measuring rates and abundances of diazotrophs provide
information on rates in samples, but given the relatively small number of these
observations and the expanse of the oceans, the extrapolation to basin scales is associated with large uncertainties. Biogeochemical proxies are measures of nutrients or
nutrient ratios that reflect gain (e.g. by N 2 fixation) or loss (e.g. by denitrification or
Anammox) of N relative to P. N* or P*, linear expressions of regeneration stoichiometry relative to the Redfield ratio of 16:1 N:P, have been used to predict regions
10.5 Measurements
The
15
N method coupled with nanoscale secondary ion mass spectrometry
(nanoSIMS) has also been used to measure cell-specific rates of N 2 fixation.
New methods continue to be developed. Since H 2 is evolved by nitrogenase, H 2
flux can be related to N 2 fixation activity. H 2 gas measurements are relatively simple
to make and can be used to obtain shipboard data in almost real time. A modified
method using
13
C-labeled acetylene called the isotopic acetylene reduction assay
(ISARA) makes it possible to measure N 2 fixation by alternative nitrogenases, as
well as that catalyzed by the Mo nitrogenase.
Since only a few of the diazotrophs can be easily identified visually, either by the
naked eye (e.g. Trichodesmium) or microscopically (e.g. Richelia), molecular biological methods have been used to identify and quantify diazotrophs in a broad
range of marine habitats. The introduction of the polymerase chain reaction (PCR)
led to a method using degenerate nitrogenase (usually nifH) primers to amplify nif
genes from uncultivated microorganisms, thereby enabling the discovery of new
organisms and establishing the true diversity of diazotrophs in the sea. More
recently, the advent of metagenomics and metatranscriptomics has provided unbiased approaches to cataloguing nif genes, although diazotrophs are generally in low
abundance. Sequence types representing specific lineages can be targeted with customized probes: using these specific probes, quantitative PCR (qPCR) has provided
much of the recent data on distributions, abundances and gene expression (RT-qPCR)
of uncultivated microorganisms including UCYN-A and NCDs.
Uncultivated diazotrophs have been visualized using fluorescent in situ hybridization (FISH) often coupled to catalyzed reporter deposition (CARD-FISH) to
enhance detection. This technique targets rRNA, thus the rRNA gene sequence must
be known, which it is not for many uncultivated diazotrophs that are only known by
their amplified nifH sequence. Also, CARD-FISH effectiveness is limited by cell
abundances: since diazotroph densities can be low it can be difficult to obtain sufficient numbers of cells on a filter to obtain good quantitative estimates.
Another approach for identifying diazotrophs termed Stable Isotope Probing
(SIP) is based on the increased density of genomic DNA labeled with
15
N. DNA is
extracted from samples incubated with enriched
15
N 2 gas, and the heavier labeled
DNA separated by ultracentrifugation. Sequencing of DNA from the heavier fractions is presumptively from diazotrophic organisms that have synthesized DNA (or
in some applications RNA) using the N from heavy labelled
15
N 2 gas. This method
is not quantitative, but does directly identify gene sequences associated with active
N 2 -fixing microorganisms, if enriched sufficiently. Several studies of marine N 2
fixation have coupled SIP with nanoSIMS which may allow for quantitative
interpretation.
The methods for directly measuring rates and abundances of diazotrophs provide
information on rates in samples, but given the relatively small number of these
observations and the expanse of the oceans, the extrapolation to basin scales is associated with large uncertainties. Biogeochemical proxies are measures of nutrients or
nutrient ratios that reflect gain (e.g. by N 2 fixation) or loss (e.g. by denitrification or
Anammox) of N relative to P. N* or P*, linear expressions of regeneration stoichiometry relative to the Redfield ratio of 16:1 N:P, have been used to predict regions
10.5 Measurements