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CHAPTER 2. HOW MUCH NITROGEN DO LEGUMES FIX?
THOMAS A. LARUE AND THOMAS G. PATTERSON
part to the ability of legumes, in symbiosis with rhizobia, to obtain nitrogen from
But how much nitrogen is obtained? The direction of research and the future
management of legumes in agriculture will require accurate knowledge of the
amounts fixed by crops in the field. If fixation is less than we now think, then we
will not achieve the expected returns of N. If fixation is as high or higher than
some reports say, we must search out the reason why some legume crops can
deplete soil N.
Recent work in plant physiology indicates that symbiotic fixation is not ‘‘free
fertilizer”; the plant must provide energy in the form of photosynthate. Whether
a legume crop “pays” for fixation with decreased yield is not yet determined.
Legumes require more phosphate fertilizer than cereals, and many are more
demanding of water. The cost of inoculant will not be negligible to cash-poor
farmers in developing countries. Does the N fixed compensate for these inputs?
Ultimately, plant breeders and agronomists will require accurate estimates of the
amount of fixation, and of its cost, to determine whether increasing fixation is
B. THEENERGYCOSTOF SYMBIOTIC
Despite apparent differences, there is a fundamental similarity between symbiotic nitrogen fixation and the industrial production of nitrogen fertilizer.
Energy is required for both methods, for thermodynamics requires this of all
possible methods of fixation. Nitrogenase requires energy in the form of ATP
and electrons. In addition, there are energy costs associated with nodule formation and maintenance, hydrogen loss, and incorporation and transport of newly
fixed N. There must also be an energy cost for using soil nitrate, but comparisons
of the two have been difficult to examine experimentally.
Silsbury (1977, 1979) estimated the respiratory burden of subterranean clover
grown under artificial light with nitrate or NZ.He calculated the growth coefficients (the fraction of net C02uptake in light associated with the synthesis of new
dry matter) and found them constant over a 50-day period. For nodulated plants
they were significantly higher (0.189) than for plants using nitrate (0.137).
Nodulated plants used 810 mg C02 for the synthesis of 1 g dry weight, while
nonnodulated plants used only 510 mg C02.
Mahon (1977, 1978) calculated the energy cost of fixation after measuring root
respiration and estimating N fixation by acetylene reduction. He compared the
respiration of plants grown on N, with similar plants treated a few days previously with ammonium nitrate. It was assumed that the respiration due to
HOW MUCH NITROGEN DO LEGUMES FIX?
growth and maintenance was the same in both populations, and that the decreased root respiration in the treated plants was associated with decreased nitrogenase activity. Mahon obtained a value of about 6.7 g C/g N fixed with
soybeans, cowpeas, Phaseolus, and peas.
Ryle and his co-workers (1979) compared growth, photosynthesis, and shoot
and root respiration of soybean, clover, and cowpea grown on N2 or nitrate under
bright light. The plants provided with nitrate grew larger. Gross photosynthesis
did nor differ in the two populations, nor did shoot respiration. The fixing plants
had respiration rates about twice those of nitrate-grown plants. Expressed as a
percentage of the gross photosynthesis, the root respiration of fixing and nonfixing plants was 22 versus 11 for soybean, 27 versus 14 for cowpea, and 34 versus
21 for clover; that is, for three species, plants fixing their nitrogen respire
11-13% more of their photosynthate.
The relationship between root respiration and N fixed varied during plant
growth. In all species it was highest (- 15 g C/g N) in young plants. Presumably,
energy was used in forming nodules. In soybean and cowpea it dropped to a
minimum of 3-5 g C/g N just before the nodules senesced during pod fill. The
average cost was 6.3 g C/g N-a figure very close to Mahon’s.
It is remarkable that the same respiratory cost, approximately 6.5 g C/g N, was
determined by two investigators using five legumes. The close similarity
amongst species suggests that it may not be easy to find significant differences in
efficiency within a single species.
There is good evidence that photosynthate supply to nodules is a major limitation to symbiotic fixation (Hardy and Havelka, 1975). The carbon cost of 6.5 g
C/g N estimated by Ryle et al. and Mahon suggests that fixation of 1 kg NH,
would cost 15-20 kg dry weight. In evaluating some of the extraordinarily high
claims for hundreds of kilograms of N per hectare from symbiotic fixation, one
should question whether the crop is capable of fixing the required carbon and
translocating it to the root.
Would a nodulated legume crop ever yield less than one obtaining all its N
from soil? The common observation on soils very low in available N is that
effective nodulation increases yield compared to uninoculated controls. In soils
of very high fertility, the soil N suppresses nodulation and fixation, and yields
are generally equal. The aforementioned experiments, however, indicate that on
soils of intermediate fertility, the energy cost of nodule formation and fixation
might lower the yield of the crop obtaining some of its N via the symbiosis,
compared to an uninoculated control.
Such a result was observed in a Brazilian soil in which soil N was not limiting
to growth. With Phaseolus cultivars there was a positive correlation of nodule
weight with plant N content but a negative correlation with grain production
(Pessanha et al., 1972). This is what is expected if fixation requires more
THOMAS A . LARUE AND THOMAS G. PATTERSON
photosynthate than nitrate utilization. We did not find similar results elsewhere in
the literature. It may be that the difference in energy cost between nitrate and N2
usage is not so great in the field as in lab experiments.
In reviews and texts there are many published tabulations of the amount of
fixation by legume crops. Most are derived from a very few sources. Two
favorites are publications by Erdman (1949) and Lyon and Bizzell(l933, 1934).
The first was an extension pamphlet promoting inoculation, and containing data
that were not substantiated by methodology. Erdman stated that his estimates had
been calculated in most cases from controlled pot experiments which when
magnified to an acre basis gave results higher than the actual figures. Unfortunately others citing his figures did not include his caveat. The study of Lyon and
Bizzell, as we will see, did not approximate the field crop condition and probably
Bums and Hardy (1975) averaged a great many published estimates to arrive at
an average figure of 140 kg N fixed per year per hectare of arable land under
legumes. Shortly thereafter, that figure was considered by a group of scientists
attending a conference on nitrogen-fixing microbes. They concluded (Burris,
1978) that a realistic figure would be half the Bums-Hardy calculation. This
reassessment, however, was apparently based as much on intuition as on new
In the literature much of the work reported on estimating fixation cannot be
extrapolated to field conditions. A very common procedure, especially when
isotopic N is used, is to grow legumes in small pots, often in the greenhouse. The
substrate is so unlike the soil and the conditions of plant growth so different from
the field that the results cannot be used to calculate fixation by crops. Many
experimenters, including those using field plots, give results only as milligrams
N per plant or percentage N of the yield or percentage plant N derived from
fixation. In the absence of information about yield per area or planting density, it
is impossible to calculate the fixed N per hectare.
The published results of fixation as a percentage of plant N may, however, serve
in estimating a realistic figure for fixation in farm crops. It is a common observation that yields in experimental or demonstration plots are much higher than the
average crop yields in the area. It is likely true that symbiotic fixation is also less
on the farm. Nodule formation and function are depressed by a variety of environmental factors-water stress, flooding, herbicides, improper fertilizer
placement, etc. It is unlikely that the percentage N from fixation will be higher
on farms than on well-tended research plots. Therefore the percentage N fixed in
HOW MUCH NITROGEN DO LEGUMES FIX?
test plots might be used in calculating a realistic upper limit on fixation in
II. METHODS OF ESTIMATING FIXATION BY CROPS
The standard procedure for nitrogen analysis is the Kjeldahl determination
(Bremner, 1965). Its major advantages are simplicity and low expense.
The simplest estimate of N fixation is by total N accumulation of the crop.
This is based on the intuitive assumption that the crop derives all its N via
symbiotic fixation. We can find no published evidence that this is ever the case
under field conditions. The many estimates based on total plant N certainly
Growth on low-fertility soils or on soils artificially impoverished in available
N is no guarantee that all N is obtained by fixation. Kohl et al. (1980) decreased
available N in a soil by the addition of 34 tonnesha of corn cobs. The nodulated
soybean neverthelessobtained 47% of its plant N from this soil, and a nomodulated
isoline yielded 2106 kg/ha of grain. These results demonstrate the ability of a
legume to scavenge soil N.
A closer approximation to N fixed may be achieved by analyzing changes in
soil N as well as that removed in the crop. The frequently cited study by Lyon
and Bizzel (1933, 1934) was such an approach. These workers placed a mixture
of 60% silty clay loam and 40% sand in outdoor frames of unstated dimensions.
Various crops were alternated or grown together for 10 years and the N in the
crops was assayed at each harvest. The N in the top 28 cm of substrate was
analyzed at the beginning and end of the trial, and the “apparent fixation of
nitrogen” was calculated on an annual basis.
There was an accumulation of soil N under clovers, alfalfa, and vetch, and a
decrease with soybeans, peas, beans, barley, rye, and oats. The clovers had an
apparent fixation averaging 166-200 kg N/ha. Alfalfa accumulated 296-330 kg
N/ha. Soybeans, peas, and beans averaged 125, 57, and 70 kg N h a annually,
respectively. Nonlegumes accreted 2 1-37 kg Nha.
This study is considered a classic for documenting the advantage to topsoil N
of proper forage-cereal rotations. However, its limitations are that the change in
soil N has based on only two estimates 10 years apart and that only the top layer
was analyzed. Legume roots may extend downwards 3 m (Weaver, 1926). The
unaccountably high “apparent fixation” by nonlegumes suggests that there was
appreciably mobilization of nutrients from below the sampled zone.
THOMAS A . LARUE AND THOMAS G . PATTERSON
Experiments on legumes growing on artificially impoverished soils are not
uncommon in the literature. The substrate used by Lyon and Bizzell was an
artificial one and for that reason the data they obtained should not be extrapolated
to estimate fixation by farm crops.
Long-Term Nitrogen Balance Studies Using Lysimeters
A lysimeter is an enclosed soil system in which the addition and removal of
nutrients, water, and plant material can be controlled and measured. Although
many designs have been described (Kohnke et al., 1940), the basic construction
consists of a tank or box placed in the ground and filled with soil. Leachate from
the soil in the tank is collected from the bottom of the lysimeter. Lysimeters have
been used in long-term studies on nitrogen balance in crops under different
management systems (Chapman et al., 1949). In addition, lysimeters can be
used to compare and calibrate different techniques for estimating N fixation
under controlled conditions (Williams et al., 1977).
Several disadvantages are inherent in lysimeter studies. They are expensive to
install because they require the excavation of large volumes of soil and must be
constructed of materials resistant to corrosion. Due to their expense, the size and
number of lysimeters, and thus the number of treatments and replications, is
In some lysimeter studies, long-term nitrogen balances have shown unaccounted losses of nitrogen (Collison et al., 1933). This is attributed to volatilization of N from the lysimeters (Chapman et al., 1949; Patwary and Raikavich,
1979), although this assumption has been challenged (Craswell and Martin,
An adjusted measure of fixation by the nitrogen accumulation technique is
obtained when the contribution of soil N to the total N of legumes is estimated.
This correction for the contribution of soil N is obtained by growing a nonfixing
plant in comparison with the N-fixing legume. Total N content of the nonfixing
crop (derived solely from soil N) is subtracted from the total N content of the
N-fixing legume. The difference between the values is assumed to be the quantity
of N derived by N fixation. This procedure is often referred to as the “difference” method (Williams et al., 1977). Three versions of the difference
method are commonly used.
1 . Comparison of a Legume with a Nonlegume
Soil N contribution to a fixing legume is estimated by growing a nonlegume
concurrently with the legume. Annual small grains such as wheat and oats have