Environmental Engineering Reference
In-Depth Information
periodically high water table which would result in significant losses of the fertilizer
N through denitrification.
In both methods, tissue samples from both species (N
2
-fixing and non-fixing) are
collected, dried, and ground. Nitrogen in the ground tissue subsamples is converted
to NH
4
+
-N through acid digestion which is then oxidized to N
2
. The N isotope ratio
of the N
2
sample is determined by mass spectrometry or emission spectrometry.
There are commercial labs that will run N isotope analyses on ground tissue
samples. The only data needed from the control species is the N isotope ratio.
To determine quantities of N fixed, biomass and total N content must be determined
for the fixing species. Because the differences between the
15
N/
14
N ratios in the
atmosphere and that found in soils are very small, there are procedural differences
between the isotope dilution method and the natural abundance method (Weaver
and Danso
1994
). First, in the natural abundance method, isotopic composition is
expressed as
15
N instead of At.%
15
N, where
15
N
δ
δ
¼
[(R
sample
R
standard
)/
15
N/(
14
N+
15
N), so that 1
15
Nunit
R
standard
]
0.00037 At.%
excess
15
N. Second, it is recommended that a member of the fixing species be
grown hydroponically on N-free medium to provide an estimate of discrimination
that occurs during N
2
fixation. Third, multiple non-fixing plant species should be
sampled to provide a mean of the isotopic composition of the soil. With the natural
abundance method, the percentage of plant N derived from fixation (%Ndfa) is
calculated as follows: %Ndfa
1,000, and R
¼
δ
¼
15
N of the
¼
(x
y)/(x
f)
100, where x
¼ δ
15
N of the fixing plant grown in soil; and f
15
N of the
non-fixing plants; y
¼ δ
¼ δ
fixing plant grown hydroponically.
7.5.3.2 Acetylene Reduction
Nitrogenase, the enzyme that reduces N
2
to NH
3
(N fixation), will also reduce
acetylene (C
2
H
2
) to ethylene (C
2
H
4
). The acetylene reduction method has been
used to provide a point in time assessment of N
2
fixation in both symbiotic systems
(e.g., nodulated legumes) and free living organisms. The following method was
presented by Carpenter et al. (
1978
) to assess N
2
fixation by free living bacteria and
algae in tidal marshes. They used surface cores taken from vegetated marsh areas to
target algae and sediment slurry samples taken from pannes to target cyanobacteria.
A plastic corer is used to take a surface core (0.5 cm
2
dia., 0.25 cm deep); a pipette
is used to remove 1 ml of slurry. Each sample is placed in a 6.5 ml wide mouth
serum bottle, the bottles are capped, and injected with 1 cc high-purity C
2
H
2
gas.
The samples are gently rotated to facilitate solution of the C
2
H
2
, and incubated for
1 h. A gas sample is than removed and analyzed for C
2
H
4
by gas chromatography.
Limitations to the acetylene reduction method include the point in time nature of
the assay and the indirect nature of the assay which makes extrapolation to the
amount of N
2
fixed questionable. Seasonal rates of N fixed cannot be determined
from a point in time measurement because the rate may vary over time. Quantifying
N
2
fixed from the amount of C
2
H
2
reduced requires a conversion factor. Hardy
et al. (
1968
) first suggested a theoretical conversion factor of 3 mol C
2
H
2
reduced to
