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Soil Science and Plant Nutrition

ISSN: 0038-0768 (Print) 1747-0765 (Online) Journal homepage: http://www.tandfonline.com/loi/tssp20

Nitrogen fixation in Azolla-Anabaena symbiosis as


affected by mineral nutrient status

Michihiko Yatazawa , Naoki Tomomatsu , Noriyo Hosoda & Katsunori


Nunome

To cite this article: Michihiko Yatazawa , Naoki Tomomatsu , Noriyo Hosoda & Katsunori Nunome
(1980) Nitrogen fixation in Azolla-Anabaena symbiosis as affected by mineral nutrient status, Soil
Science and Plant Nutrition, 26:3, 415-426, DOI: 10.1080/00380768.1980.10431227

To link to this article: http://dx.doi.org/10.1080/00380768.1980.10431227

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Soil Sci. Plant Nutr., 26 (3), 415-426, 1980

NITROGEN FIXATION IN AZOLLA-ANABAENA


SYMBIOSIS AS AFFECTED BY MINERAL
NUTRIENT STATUS

Michihiko YATAZAWA, Naoki TOMOMATSU,* Noriyo HosoDA,


and Katsunori NUNOME**
Department of Agricultural Chemistry. Nagoya University.
Nagoya. 464 Japan

Received December 20, 1979

The growth and nitrogen-fixing capacity of Azalia imbricata were studied with special
reference to the effects of the mineral nutrient status in the medium. The threshold levels of
P, K, Mg, and Ca in the medium for Azalia growth were ca. 0.03, 0.4, 0.4, ~nd 0.5 mmol'liter-t,
respectively. Full development of nitrogenase activity was not realized at concentrations below
0.03, 0.6, 0.5, and 0.5 mmolliter-t, respectively. Combined nitrogen was unfavorable for
growth and nitrogen fixation. Deficiency of anyone of the micronutrient elements, Fe, Mn,
Co, Zn. Cu, Mo, and B, had an unfavorable effect on growth and developing nitrogenase
activity. The threshold levels of Fe, Mn, Mo, and B for growth were 50, 20, 0.3, and 30
,ugliter-" and those for nitrogenase activity were 20,10, I, and 20 ,ugliter- l, respectively. Fe
and Mo were very sensitive for developing nitrogenase activity. On the other hand, B was
shown to affect growth specifically.
The nitrogen-fixing capacity measured from the increase in total nitrogen in Azalia plants
cultured with complete nutrient solution lacking only combined nitrogen, was 6.50 mg N .g-l
(D.W.). day-I. On the other hand. the acetylene-reducing capacity of the plants was 34.6 ,umole
~H, producedg- l (D.W.)hr-l. These two values are not so different from one another.
The very low nitrogen-fixing capacities reported by pioneer workers were found to be raised
considerably when the data were recalculated on the basis of the daily increase ratio in growth.
Illumination (up to 6,000 lux) of the plants during the period of acetylene reduction, greatly
enhanced nitrogen fixation. This effect is discussed in relation to the saturation point in
photosynthesis.
Additional Index Words: nitrogen fixation, Azalia.

Nitrogen fixation in Azalia-Anabaena symbiosis has long been known from the
work of several pioneer investigators (2, 5, 12, 15). The host plant Azalia occurs
widely on the surface of bodies of fresh water in the tropical and temperate regions.
About six extant species are known globally. Considerable numbers of practical
experiments have been reported on this symbiosis based on its potential importance in
agriculture (4, 11, 16, 17). However, quantitative determinations of the nitrogen-
Present address: Yakult Honsha Co., Ltd., Fujisawa Factory.
Present address: Bureau of Public Health, Tokyo Metropolitan Government.
415
416 M. YATAZAWA, N. TOMOMATSU, N. HOSODA, and K. NUNOME

fixing activity in the symbiosis are rather limited (1, 3, 12, 15-17), and information
concerning the levels of mineral nutrients required for sufficient display of nitrogen
fixation in the symbiosis is almost lacking.
This paper reports quantitative relations between mineral nutrient status and
nitrogen-fixing capacity in Azolla imbricata-Anabaena symbiosis measured by acetylene
reduction assay and the Kjeldahl analytical method.

MATERIALS AND METHODS

Azolla. The Azolla plants employed were originally collected from a pond in
Chita, Aichi Pref., Japan. The Azolla was identified as Azolla imbricata (13) associated
with Anabaena azollae. A. imbricata is sometimes used as a synonym of A. pinnata.
However, Japanese taxonomists always classify it as a different species from A. pinnata
(13).
Culture methods. Several plant colonies were taken from a stock culture in a
small pond and precultured with a dilute complete nutrient solution lacking only
combined nitrogen for a month. Precultured ex plants of about 50 mg fresh weight
were incubated with 200 ml of nitrogen-free nutrient solution (AII( - N) or its modi-
fied nutrient-deficient culture solutions. The AII( - N) solution contained the fol-
lowing macro nutrients (in mmoHiter- 1): NaH 2 PO, 1.00, K 2SO, 2.00, CaCI 2 3.00, and
MgSO, 2.00; plus the following micronutrients (in pmoHiter- 1): FeSO,o7H 20 4.33,
MnSO,o4H 20 1.13, CuSO,'5H 20 0.08, ZnSO,o7H 20 0.19, NaMo0 4 H 20 0.05, and
HaBOa 5.77. Nutrient-deficient solutions were prepared by reducing one component
to various levels from that in the AII( - N) solution. The chemicals used were of
analytical grade, and no special purification of them was performed. The water for
making the culture solutions was prepared by passing glass distilled water through ion-
exchange resins. The pH of each solution was adjusted to 6.5. The culture vessels
were 500 ml Tystone glass beakers except in the case of B-deficient culture experiments,
where polyethylene beakers were used. They were covered with glass or plastic trans-
parent dishes to avoid mineral contamination from dust materials. They were placed
on a desk in a phytotron with 12 hr light at 30C and 12 hr darkness at 25C. The
light intensity was 4,000 lux at the surface of the desk unless otherwise indicated. The
culture period was 3 weeks unless otherwise indicated.
Measurement of growth. Harvested plants were placed on a dry filter paper to
remove adhering culture solution. The fresh weight was measured, and the growth
value (G.V. = fresh weight of harvested plants/fresh weight of initial explants) was
calculated. The daily increase ratio (R) was also calculated from the equation, (I +
R)t=G.V., where t is the period (day) of the culture.
Measurement for nitrogenase activity. Harvested plants weighing about 0.3 g
(F.W.) were placed in a 50-ml conical flask under light of 8,000 lux. The amount of
C 2H, produced in an atmosphere containing 10% C2 H 2 during an incubation period
of 150 min, was measured with a gas-chromatograph (Shimadzu GC-4CPF) under the
Nitrogen Fixation in Azolla-Anabaena Symbiosis 417

same conditions as described previously (7). After chromatographic measurement,


the plant samples were dried and weighed. Nitrogenase activity was expressed in most
cases in pmole C2H4 produced.g-1(D.W.)hr- 1.
Analysis of combined nitrogen. The total nitrogen of the dried plant materials and
of the culture solution was measured by the Kjeldahl method. Ammonium-N, nitrate-
N, and nitrite-N were analyzed by standard methods (6).

RESULTS

Nitrogenase activity under different light intensities


Since light affords energy for nitrogen fixation, the effect of light intensity on the
acetylene-reducing activity of Azolla was examined by exposing the cultures to different
light intensities during the period of incubation with acetylene. As shown in Fig. 1,
illumination of the plants during the period of acetylene reduction greatly enhanced
the nitrogenase activity in the plants, although acetylene reduction was still recognized
even in the dark, presumably due to the utilization of reserved carbohydrates which
had been synthesized in daytime.

Effects of macronutrient elements


The growth and nitrogenase activity of Azolla plants cultured with macronutrient-
deficient solutions for 21 days at the first transference are shown in Table 1. Inocu-
lated explants on Ca- or Mg-deficient solution grew only to a slight extent. On the
other hand, considerable growth was obtained with P- or K-deficient plants. This
was thought to be due to carry-over of P and K with the inoculated explants. To
obtain more accurate data for the effects of macronutrient elements in the medium on
growth and nitrogenase activity in Azolla, P- or K-deficient plants obtained at harvest
of the first transference culture were reinoculated into modified AII( - N) solution

100 o
.....o
-c;::: 80
'"03:.
Eo
::::- 60
>1
.- bO
~cU
~o 40
-oE
CI) ::t.

~~
E N
20
"'U
<.J
<.J
c(
90 120 150
Period of inCUbation (min)
Fig. 1. Nitrogenase activity in Azolla-Anabaena symbiosis as
affected by light intensity.
418 M. YATAZAWA, N. TOMOMATSU, N. HOSODA, and K. NUNOME

Table 1. Growth and nitrogenase activity in Azolla-Anabaena associates cultured


with macronutrient-deficient solution.

Culture solution Growth % of Nitrogenase activity % of


value control (pmole C1H,g-1 (D.W.)hr-l) control

All ( - N): control 20.4 100 33.5 100


All (-N)-P 13.3 65 11. 3 34
All (-N)-K 14.0 69 13.7 41
All (-N)-Ca 0.8 4 0.0 0
All (-N)-Mg 4.5 22 0.2 0.6

Mean of 4 replicates.

Nitrogenase activity
-O-P
.... K 1.0
-A-Ca 1.0
-x- Mg .::-:;;
0.8 .s= 0.8 'B.,
i .,
0.6 ;;;, 0.6 ~
~ gj,
o
0.4 ~ ., 0.4 !:
'c
0:: .,
0.2 0.2 ~
Qj
0::
'::----,,----'-----:----'=""'-:---'-----'0.0
10
Concentration of macronutrients (mmole) Concentration of macronutrients (mmole)
Fig. 2. Growth and nitrogenase activity in Azolla-Anabaena associates as affected by the level of
macronutrients in the culture solution.

containing various levels of P or K. On the other hand, fresh explants were cultured
with other modified All( - N) solutions containing various levels of Ca or Mg. The
results showed that elimination of carry-over of P or K resulted in sufficient reduction
of growth and nitrogenase activity in the plants (Fig. 2). The threshold levels of P,
K, Mg, and Ca for Azolla growth were found to be about 0.03, 0.4, 0.4, and 0.5 mmol
liter-I, respectively. Below the threshold value, the decrease of growth was very sharp
with Ca-deficient plants. The nitrogenase activity of Azolla decreased sharply through
the reduction of macronutrient elements. Full development of nitrogenase activity
was not realized at concentrations below 0.03, 0.6, 0.5, and 0.5 mmolliter- 1 for P,
K, Mg, and Ca, respectively.
As regards deficiency symptoms, plants deficient in P became slightly pale, and
the roots elongated extraordinarily. Necrosis on the fronds was also recognized in
plants deficient in K, Ca, or Mg. Plant colonies deficeint in Mg were liable to dis-
integrate into small colonies.
The effects of combined nitrogen were examined by supplementing ammonium
Nitrogen Fixation in AzalIa-Anabaena Symbiosis 419

sulfate to AU( - N) solution at various levels of nitrogen. A considerable decrease


in growth value was observed at higher concentrations of combined nitrogen (Table
2). Under these conditions, green algae grew intensely on the surface of the culture
solutions. The nitrogenase activity was also decreased to some extent.

Effects of micronutrient elements


Azolla plants were cultured with micronutrient-deficient solutions for 21 days.
The growth and nitrogenase activity of the harvested plants were measured (Table
3A). At the first transference, the Azolla plants grew reasonably well except for B-
and Fe-deficient plants. This was thought to be due to carry-over of micronutrient
elements from the inoculated explants and by contaminants from the reagents and
water employed. On the other hand, the nitrogenase activity decreased considerably
in plants deficient in Fe, Mo, and B in that order. To examine the comparative im-
portance of each micronutrient element in the development of nitrogenase activity and
the amount of growth, ratios for relative nitrogenase activity/relative growth value
were calculated (Table 3). The data suggest that Fe and Mo are characteristically
sensitive for developing nitrogenase activity, while B may affect growth rather specifi-
cally.
In an attempt to reduce the carry-over of micronutrient elements from the inocu-
lating explants, part of the harvested plants of the first transference (Table 3A) was
reinoculated into each micronutrient-deficient culture solution. After 21 days incuba-
tion, the growth and nitrogenase activity were measured (Table 3B). Further decreases
in growth value and specific nitrogenase activity were obtained for plants cultured
with Fe-, B-, Mo-, and Mn-deficient solutions. Other plants grew similarly to the
first transference plants. This may have been due to inevitable contamination of these
micronutrient elements from the culture medium and explants.
To obtain information on the threshold levels of Fe, Mn, B, and Mo in the medium
for the growth and nitrogenase activity of Azolla, explants which had been slightly
deficient in each of the micronutrient elements were reinoculated into culture solutions

Table 2. Effect of combined nitrogen (NH,-N) on the growth and nitrogenase activity
in Azalia-Anabaena associates.

Culture solution Growth %of Nitrogenase activity % of


value control (pmole C 2 H, produced g-l (D.W.). he ' ) control
All (-N): control 15.8 100 36.9 100
All (-NHN 2 ppm 11. 1 70 29.8 81
All (-NHN 5 ppm 10.6 67 30.5 83
All (-N)+N 10 ppm 10.7 68 24.8 67
All (-N)+N 20 ppm 9.0 57 24.6 67
All (-NHN 40 ppm 6.8 43 22.2 60

Mean of duplicate.
420 M. YATAZAWA, N. TOMOMATSU, N. HOSODA, and K. NUNOME

Table 3. Growth and nitrogenase activity in Azolla-Anabaena associates cultured


with micronutrient-deficient solution.
A. First transference culture

Growth % of Nitrogenase activity % of


Culture solution value control (pmole C,H, produced. g-1 control (B)/(A)
(A) (D.W.)hr-l) (B)
- --~---~~-~

All ( - N): control 20.4 100 33.5 100 1. 00


All (-N)-Fe 12. 1 59 9.23 28 0.47
All (-N)-Mn 16.4 80 27.7 83 1.04
All (-N)-Co 16.0 78 26.3 79 1. 01
All (-N)-Zn 16.4 80 26.2 78 0.98
All (-N)-Cu 16.3 80 22.4 67 0.84
All (-N)-Mo 15.3 75 13.1 39 0.52
All (-N)-B 6.22 30 13.5 40 1. 33

B. Second transference culture

Growth % of Nitrogenase activity %of


Culture solution control (pmo)e C 2H, produced. g-1 control (B)/(A)
value (A) (D.W.) hr- ' ) (B)

All ( - N): control 19.2 100 32.2 100 1.00


All (-N)-Fe 4.9 26 2.0 6 0.23
All (-N)-Mn 11. 1 58 11. 9 37 0.64
All (-N)-Co 14.8 77 25.0 78 1. 01
All (-N)-Zn 14.8 77 19.2 60 O. 78
All (-N)-Cu 15.8 82 19.3 60 O. 73
All (-N)-Mo 12.8 67 10.7 33 0.49
All (-N)-B 4.8 25 10.3 32 1. 28

Mean of 4 replicates.

containing various levels of micronutrient elements. After 21 days incubation, the


growth and nitrogenase activity were measured. The results indicated that, for nitro-
gen-fixing activity, the threshold levels of Fe, Mn, Mo, and B in the culture solution
were roughly 20, 10, 1, and 20 pgliter- 1, respectively, and the levels for growth were
about 50, 20, 0.3, and 30 pgliter-t, respectively, under the experimental conditions
employed.
During the course of the culture, chlorotic symptoms appeared in Fe-deficient
plants. Newly developed fronds became strongly discolored. Iron also showed
another characteristic response in Azolla nutrition. Fe-deficient plants obtained after
19 days culture were transferred to Fe3 + solution, which was prepared by replacing
Fe2+ with Fe3 + in All( - N) culture solution, at pH 4.0 or 7.0 and incubated for 17
days. Plants grown at pH 4.0 became dark green and the growth value was restored
Nitrogen Fixation in Azolla-Anabaena Symbiosis 421

to 10.6 and the acetylene-reducing capacity to 36,umole C 2H 4 produced "g-I(D.W.) "


hr- l . On the other hand, plants grown at pH 7.0 showed intensified deficiency symp-
toms and gave a smaller growth value of 4.2 and a lower nitrogenase activity of 3.2
,umole C2H 4 produced" g-I(D.W.)" hr-l.

Nitrogen-fixing capacity as estimated from the nitrogenase activity


The nitrogenase activity in plants grown on AII( - N) culture solution was
measured with 31 cultures. The values obtained ranged from 25 to 45 ,umole C 2H 4
produced" g-I(D.W.)" hr- l (average 34.6), with a light intensity of 8,000 lux during the
period of acetylene reduction" Assuming that the conversion ratio for C2H 4 produced:
N2 reduced is 3.0, the day length is 12 hr, the average light intensity in daytime is 8,000
lux, and the nitrogen-fixing activity in the dark is 10% of that in daytime, then the
nitrogen-fixing capacity in Azolla imbricata calculates at 4.26 mg N"g-I(D.W.)"day-l.

Nitrogen-fixing capacity estimated from the increase in total nitrogen


Confirmatory evidence of nitrogen fixation can be obtained from the increase in

Table 4. Nitrogen-fixing capacity of Azolla-Anabaena associates as determined


from the increase in total nitrogen in the culture.

Plant wt. per culture


Culture
period in Explants Harvested Growth
Exp. No. days F.W. value
(1 )
(mg) F.W. D.W.
(mg) (mg)

15 50 653 36.6 13.1


2 21 31 512 29.2 16.5
3 17 43 671 37.6 15.6
4 20 40 499 28.9 12.5
5 21 31 512 28.2 16.5
6 22 32 582 32.6 18.2
Total N in N concentration
Daily harvested in harvested N-fixing capacity
increase crops crops (D) (RxD)
ratio (R) (mg) (mg Ng-l (D.w. (mg Ng-l (D.W.).day-l)

0.187 1. 53 41. 8 7.82


o. 143 1. 20 41.1 5.88
0.175 1. 61 42.8 7.49
0.135 1.19 41. 2 5.56
O. 143 1. 23 43.6 6.23
0.141 1. 39 42.6 6.01
Aver. O. 154 42.2 6.50
)0S:(ifowth value)
R: (I +R)t = growth value. R = 10 t -1.
422 M. YATAZAWA, N. TOMOMATSU, N. HOSODA, and K. NUNOME

total nitrogen of the culture. To evaluate the results obtained by the acetylene reduc-
tion method, therefore, the amount of Kjeldahl nitrogen in the cultures was measured.
A few previous papers (5, 15) have reported that considerable amounts of combined
nitrogen were found in culture solutions of Azo/la pinnata. Several residual culture
solutions after the harvest of crops grown with All( - N) solution were therefore
analyzed for ammonia, nitrate, and nitrite. No indication of the presence of ammonia,
nitrate, or nitrite in the culture solutions was found under the experimental conditions
employed. Kjeldahl analysis of these culture solutions also gave only negligible
amounts of combined nitrogen.
Table 4 gives some examples of the nitrogen-fixing capacity as measured by the
Kjeldahl method. The nitrogen-fixing capacity indicated by the increase in mg N
g-l(D.W.)day-l was calculated by mUltiplying the nitrogen content of the harvested
plant~ in mg Ng-l(D.W.) by the daily increase ratio in growth on a dry matter basis.
This daily increase ratio on a dry matter basis was assumed to be the same as that of
the daily increase ratio on a fresh weight basis (R), since values for dry matter of the
explants were not available. In the present study, the R value was found to be within
the range of 0.141 to 0.187 (average 0.154). The nitrogen content in the harvested
crops was 42.2 mg Ng-l(D.W.) on average. The nitrogen-fixing capacity in Azolla
imbricata-Anabaena associations cultured with AII( - N) solution for various periods
of incubation therefore lay within the range of 5.56-7.82 mg N .g-l(D.W.)day-l (aver-
age 6.50) under the experimental conditions employed.

DISCUSSION

Culture solution. Various kinds of culture solutions have been used by previous
authors. The composition of the culture solution in the present study was basically
the same as that of the medium used by JOHNSON et al. (9) with only small amendments.
The growth of Azolla with AII( - N) culture solution was reasonable (Table 4), but
a somewhat lower concentration of macronutrient elements would be preferable under
the present experimental conditions, especially the light intensity employed. The daily
increase ratio of 0.154 corresponds roughly to 116 hr as doubling time. The magni-
tude of the value was about the same as those obtained by other workers. The dou-
bling time estimated by BROTONEGORO and ABDULKADIR (3) was 63-105 hr, and that of
WATANABE et al. (17) was 3-5 days. The daily increase ratios calculated from the data
of TUZIMURA et al.(J6) and JOHNSON et al. (9) were 0.179 and 0.177, respectively.
The slightly lower value in the present experiment may be attributable to the lower
light intensity during the incubation period.
Light intensity as affecting the nitrogenase activity. The present results clearly
indicated that there is a positive effect of light intensity in enhancing the nitrogenase
activity in Azolla plants (Fig. 1). This finding was seemingly different from Broto-
negoro's results, in which nitrogenase activity was not reduced under a 65% reduction
of full sunlight for 5 hr, even though further reduction of the light intensity led to a
Nitrogen Fixation in Azolla-Anabaena Symbiosis 423

decrease in nitrogenase activity. In their experiment, the light intensity of full sun-
light was about (2-6) x 104 lux. More than 7,000 lux would thus have been supplied
to the plants under 65% reduction of full sunlight. The results of the present study
were obtained under light intensities below 6,000 lux. The combined experimental
data suggest therefore that there may be two phases of nitrogen fixation in response
to light intensity: one would proceed under a higher light intensity than the saturation
intensity above which no increase in nitrogen fixation would be expected, and the
other would operate at a lower light intensity under which a proportional increase in
nitrogen fixation would occur with increasing light intensity. The saturation intensity
would lie at about 1 x 104 lux. Inside the Azolla plant, the saturation intensity would
take a somewhat smaller value than that. The value, in fact, coincides well with the
light saturation point in photosynthesis usually observed in shade plants. The situa-
tion of Anabaena azollae in cavities of the Azolla leaf appears to be similar for getting
light as that of such common shade plants, and consequently Anabaena azo/lae can be
expected to have a similar value of light saturation point in photosynthesis as that
observed in these shade plants. This, in turn, suggests that nitrogen fixation in Azolla-
Anabaena symbiosis operates principally by being supplied with reducing energy di-
rectly from the algae, at least under light conditions.
The smaller, but significantly positive capacity for nitrogen fixation observed in
Azolla in the dark is presumably supported by photosynthates supplied from the algae
themselves or their host plant Azolla, as has been postulated by PETERS (14).
Effects of macronutrient elements. The importance of macronutrient elements
including P, K, Ca, and Mg in supporting the growth of Azolla has been universally
recognized by previous authors (16, 17). However, the degree of reduction in growth
of plants cultured with macronutrient-deficient solutions varies with different authors
(16, 17). The reasons for this variation may derive mostly from differences in pre-
culture conditions, since further reduction of carry-over nutrients from the explant
should surely result in a smaller yield of harvested crops. This was clearly shown in
the present experiment in which Azolla was cultured repeatedly with a macronutrient-
deficient solution (Fig. 2). The degree of reduction in growth together with that in
nitrogenase activity of plants deficient in a particular nutrient element thus tells only
the liability of establishing deficient conditions with the particular element. In this
sense, Ca under certain natural freshwater environmental conditions, would represent
the first among the four macronutrients to become short for Azolla.
Combined nitrogen was unfavorable for growth and nitrogen fixation in Azo/la
(Table 2). Similar effects have been reported by other authors (3,10,12,17). although
somewhat contradictory favorable effects have been described by a few (2, 16). The
unfavorable effect appears to be due mainly to the vigorous growth of green algae
under nitrogen-rich conditions.
Effects of micronl!trient elements. Very few papers have dealt with the effects
of micronutrient elements on the growth and nitrogen-fixing capacity of Azolla. Avail-
able information is confined to the requirements for the following elements: Fe (10,
424 M. YATAZAWA, N. TOMOMATSU, N. HOSODA, and K. NUNOME

16, 17), Co (9), and Mo (2, 10, 16). In the present experiment, deficiency of the micro-
nutrient elements, Fe, Mn, Co, Zn, Cu, Mo, and B, produced unfavorable effects on
growth and nitrogen fixation in Azolla. Repeated transfer of the culture to micro-
nutrient-deficient solution increased the degree of depression of both activities. How-
ever, the depression in some micronutrient-deficient plants was sustained at a definite
level, presumably due to the unavoidable contamination of micronutrient elements
from reagents and water under the present experimental conditions. Fe, B, Mo, and
Mn were more liable to become deficient. The threshold levels of these elements for
the growth of Azolla were almost the same as those found for common angiosperm
crop plants (8).
Quantitative value of nitrogen-fixing capacity in the symbiosis. Several papers have
dealt quantitatively with the nitrogen-fixing capacity in Azolla-Anabaena symbiosis.
The first determination was made by OES (12) using the Kjeldahl method. The nitro-
gen-fixing capacity was recalculated by us from his data, in which the daily increase
ratio of Azolla in his 11 experiments was 0.59 on average and the N content was 2.84%
on a D.W. basis. The capacity was so estimated at about 1.7 mg N 'g-l(D.W.)day-l.
This value is more than 4 times that given by MooRE (11), who may have made calcu-
lations using a simple mean of the increased nitrogen. SAUBERT'S results (15) indicated
a nitrogen-fixing capacity in Azolla pinnata of about 2.67 mg Ng-l(D.W.)day-l,
when recalculated on the basis of a mean daily increase ratio of 0.082 and a mean N
content of 3.24% on a D.W. basis. In his experiment, a certain amount of combined
nitrogen was released into the culture solution. When a recalculation was made to
include the released nitrogen, the nitrogen-fixing capacity of Azolla increased to 3.72
mg N 'g-l(D.W.)day-l. These values also differ greatly from that referred to by
MOORE (11) for Saubert's results. Moore himself obtained direct evidence to suggest
a nitrogen-fixing capacity of 222 pg Ng-l(D.W.)day-l in Azolla pinnata when he
applied 15N excess dinitrogen to Azolla-Anabaena symbiosis for 19 days. His value
would be increased by a recalculation made on the basis of the daily increase ratio in
growth. In the present experiment, the nitrogen-fixing capacity of Azolla imbricata
was estimated to be 6.50 mg Ng-l(D.W.)day-l on averge. This value is consider-
ably larger than those obtained by the above pioneer workers.
On the other hand, the nitrogen-fixing capacity determined by the acetylene reduc-
tion method has been reported to take somewhat larger values. According to BECKING
(1), Azolla pinnata reduced acetylene at a rate of 3-6 nmole'mg- 1 protein'min- 1 corre-
sponding to 36-72 pmole C 2H, producedg-1(D.W.)hr- 1. BROTONEGORO and ABDUL-
KADIR (3) reported that Azolla pinnata fixed dinitrogen at a rate of 60-90 pmole C 2 H,
producedg-l(D.W.)hr-l in daytime, amounting to as much as 7.2 mg Ng-l(D.W.)
day-I. WATANABE et al. (17) reported that Azolla fixed 340 102 nmole C 2H, produced
g-l(D.W.)hr- l . If the moisture content of Azolla was 94%, this value would corre-
spond to 5.7 pm ole CaH4 producedg-l(D.W.).hrl.
In our case, the acetylene-reducing capacity of Azolla imbricata cultured with All
(- N) solution was 34.6 pmole' g-l(D.W.) hr- I as an average of 31 determinations.
Nitrogen Fixation in Azolla-Anabaena Symbiosis 425

This value is somewhat smaller than those obtained by BECKING (1) and BROTONEGORO
and ABDULKADIR (3), and the difference is presumably attributable to the different
experimental conditions, especially the use of a much lower light intensity during
incubation. The value of 34.6,umole C2H, produced.g-1(D.W.).hr- 1 would corre-
spond to 4.26 mg N2 fixedg-1(D.W.)day-l as mentioned above, and is not so differ-
ent from the value of 6.50 mg N .g-l(D.W.)day-l determined by the Kjeldahl method
in the present study.
Very large variations in nitrogen-fixing capacity have sometimes been noted be-
tween the results of the pioneer workers as referred to by MOORE (11) and results which
have been published recently. Such variations may well be cancelled when the data
of the pioneer workers are recalculated on the basis of the daily increase ratio as men-
tioned above.
Acknowledgements. Thanks are due to Mr. S. Hamashima, Takakura High School, Nagoya, for
his gift of Azolla imbricata plants, and to Dr. I. Watanabe, IRRI, Philippines, for his kind provision of
information on recent studies. This research was supported in part by a grant from the Ministry
of Education, Science and Culture of Japan.

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