APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1979, p.

916-921

Vol. 38, No. 5

0099-2240/79/11-0916/06$02.00/00

Effect of Interactions Among Algae on Nitrogen Fixation by

Blue-Green Algae (Cyanobacteria) in Flooded Soils
JOHN T. WILSON,t SARAH GREENE, AND MARTIN ALEXANDER*
Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York 14853
Received for publication 20 August 1979

Nitrogen fixation (C2H2 reduction) by algae in flooded soil was limited by

interactions within the algal community. Nitrogen fixation by either indigenous
algae or Tolypothrix tenuis was reduced severalfold by a dense suspension of the
green alga Nephrocytium sp. Similarly, interactions between the nitrogen-fixing
alga (cyanobacterium) Aulosira 68 and natural densities of indigenous algae
limited nitrogen-fuing activity in one of two soils examined. This was demonstrated by developing a variant of Aulosira 68 that was resistant to the herbicide
simetryne at concentrations that prevented development of indigenous algae.
More nitrogen was fixed by the resistant variant in flooded soil containing
herbicide than was fixed in herbicide-free soil by either the indigenous algae or
indigenous algae plus the parent strain of Aulosira. Interference from indigenous
algae may hamper the development of nitrogen-fixing algae introduced into rice
fields in attempts to increase biological nitrogen fixation.

Rice supplies at least one-half of the food

supply for one-third of mankind (2). Improved
varieties of rice, the application of chemical fertilizers, and the use of pesticides have increased
rice production severalfold in many parts of Asia
(15). However, these gains have been threatened
by recent increases in the price of nitrogen fertilizer, resulting in a renewed interest in biological nitrogen fixation in flooded soils.
Nitrogen-fixing blue-green algae (cyanobacteria) have been considered important sources
of fixed nitrogen in flooded soils for many years
(13). De and Mandal (6) estimated that lightdependent nitrogen fixation in six soils ranged
from 15 to 45 kg of nitrogen per ha in 6 weeks,
and Singh (13) reported that Aulosira fertilissima can fix up to 53 kg of nitrogen per ha in
one season. Algae also can make significant contributions to the growth of rice. Watanabe et al.
(18) reported that inoculation of rice soils with
Tolypothrix tenuis increased grain yields 15 to
25%. De (5) found that after five crops the yield
of rice from soil exposed to the sun was twice
the yield from soil that was shaded to prevent
the growth of algae and other phototrophs.
The factors that limit the growth and activity
of nitrogen-fixing algae in soil are poorly understood. The effect of pH and the requirement of
the algae for phosphate have received attention
(5-7), but other potential factors have generally
been ignored. Interactions with other algae may
be one of the most significant ecological factors.

Hirano et al. (8) found that inocula of T. tenuis

could not colonize flooded rice fields unless the
indigenous green algae were suppressed with
CUSO4. Similarly, Watanabe et al. (17) reported
that failure of T. tenuis to colonize flooded rice
soils was associated with blooms of unicellular
green algae, although they attributed the failure
to grazing by microcrustaceans.
The purpose of this study was to determine
whether the development and activity of nitrogen-fixing algae in flooded soil are significantly
influenced by other algae and to assess the feasibility of stimulating nitrogen-fixing algae by
suppressing the other algae.
MATERIALS AND METHODS
Aulosira 68 was the gift of G. S. Venkataraman,

t Present address: Robert S. Kerr Environmental Research

Laboratory, Ada, OK 74820.

916

Indian Agricultural Research Institute, New Delhi. T.

tenuis 1482/3a was from the Culture Centre of Algae
and Protozoa, Cambridge, England. Both cultures
were unialgal but not axenic. A sample of Moreland
clay, a soil that had been cultivated to lowland rice,
was flooded with distilled water amended with 100,ug
of KH2PO4-P per ml and incubated in the light. A
portion of the green algal bloom that developed rapidly in this floodwater was transferred to Bold's medium (3) and incubated at 24C under 100 microEinsteins per s per m2 of light from fluorescent lamps.
After several successive transfers in Bold's medium,
the culture contained almost only Nephrocytium sp.
To prepare inocula for the flooded soils, cultures in
the late exponential phase of growth were collected by
centrifugation at 4C, and the cells were washed with
distilled water and suspended in either distilled or tap
water.
Simetryne (100.0% pure) was provided by Ciba-

VOL. 38, 1979

NITROGEN FIXATION BY BLUE-GREEN ALGAE

Geigy, Greensboro, N.C. N-Methyl-N'-nitro-N-nitrosoguanidine (97% pure) was obtained from Aldrich
Chemical Co., Metuchen, N.J. Ethylene (CP) and
purified acetylene were purchased from Matheson Gas
Products, East Rutherford, N.J.
Chlorophyll was determined by fluorescence (1),
using a Turner model 110 fluorometer (G. K. Turner
Assoc., Palo Alto, Calif.); the fluorescence was standardized against an extract of T. tenuis made with a
mixture (9:1) of dimethyl sulfoxide and water (12).
The concentration of chlorophyll was determined by
the trichromatic method (1) after establishing that the
fluorescence of extracts made with dimethyl sulfoxide
and acetone were comparable at wavelengths of 665,
645, and 630 nm. To determine chlorophyll in soil, the
soil and floodwater were mixed in a Waring blender
for 120 s, and 1.0 ml of the suspension was then added
to 9.0 ml of dimethyl sulfoxide. After 2 days at 4C,
the extract was clarified by centrifugation, and the
fluorescence was determined immediately. Chlorophyll development in soils was corrected for fluorescent materials present in the soil before development
of algae.
Protein was determined by the Lowry method (4)
with 5x recrystallized egg albumin as a standard.
Acetylene and ethylene were determined by using a
Perkin-Elmer 3920B gas chromatograph fitted with a
1.0-m column packed with 2.0 g of phenyl isocyanate/
Porosil C (Waters Assoc., Milford, Mass.). The column
was operated at either 2 or 24C.
Acetylene reduction activity was determined in the
same containers used to culture the algae. Soil was
added to 500-ml polystyrene Nestrite containers
(Lilly-Tulip Division, Owens-Illinois, Toledo, Ohio).
The soil, which had an exposed surface area of 80 cm2
and a depth of approximately 1.0 cm, was flooded to
a depth of 2.5 cm. The containers were incubated at
24 to 28C under 250 to 500 microEinsteins per s per
m2 of fluorescent light from wide-spectrum Gro-Lux
lamps. The containers were arranged under the lamps
in a randomized block design, and all treatments were
replicated four times. After the appropriate interval,
each container was fitted with a transparent crystalline-polystyrene lid (Lilly-Tulip Division, Owens-Illinois) containing a rubber septum. The lids were sealed
to the containers with plastic denture adhesive (Orafix;
Norcliff Laboratories, Fairfield, Conn.). Acetylene gas
and a solution of acetylene in water were added
through the septa with a syringe. The measured acetylene concentration in the headspace and floodwater
ranged from 0.07 to 0.09 atm (ca. 7.1 to 9.1 kPa). After
8.0 h of incubation, the quantity of ethylene in the
headspace and floodwater was determined by withdrawing a 2.0-ml sample of headspace or floodwater
from the container with a syringe, injecting the sample
into a sealed, 22-ml culture tube filled with air, mixing
the contents of the tube until the gases were equilibrated, and then injecting gas from the culture tube
into the gas chromatograph. Acetylene reduction activity in the containers was linear for at least 8 h.
The tests selected for statistical analysis of the
acetylene reduction data assume a normal distribution
of the residuals (the deviations of individual replicates
from their treatment means). The data were strongly
leptokurtic; i.e., there were more residuals with low or

917

high absolute values and fewer residuals with mediumsized absolute values than would be expected from a
normal distribution. However, a logarithmic transformation of the data produced a distribution of residuals
acceptably close to the normal distribution. Therefore,
the calculation of means, the fitting of confidence
intervals, and the analyses of variance were done on a
logarithmic transformation of the data.
Samples of Moreland clay and Crowley silt loam
were obtained from rice fields near Crowley, La. Samples of Lima silt loam were collected near Aurora,
N.Y.

RESULTS
Effect of inoculated green algae. Twelve
soils were examined, but none spontaneously
produced blooms of green algae, although such
blooms are common in rice fields (13). The soils
are described elsewhere (19). The addition of
fixed nitrogen to stimulate green algae would
have inhibited the nitrogen-fixing activity of the
blue-green algae, making it impossible to follow
their development with the acetylene reduction
assay. To create a dense bloom of green algae,
therefore, a suspension of washed cells of green
algae was added to soil. The containers received
75 g of moist Lina soil (oven-dried weight, 59
g), 210 ml of distilled water, KH2PO4 (100 ,ug of
P per ml of floodwater), and ferric iron complexed with ethylenediaminetetraacetic acid (5
,ug of Fe3" per ml of floodwater). Certain containers received an inoculum of T. tenuis (0.17
mg, dry weight) or a mixed suspension of green
algae containing more than 99% Nephrocytium
sp. (3.8 mg, dry weight). In addition to Nephrocytium sp., the culture of green algae originally
contained a filamentous green alga and protozoa,
the latter apparently feeding on bacteria, but
neither the filamentous green alga nor the protozoa developed in the flooded soil.
Chlorophyll in soil inoculated with Nephrocytium sp. reached a high concentration by the
end of day 1, then growth slowed markedly
(Table 1). The chlorophyll concentration in the
soil was much greater than the maximum chlorophyll concentration achieved in culture. In soil
inoculated wtih T. tenuis alone, the chlorophyll
level continued to increase until the concentration was higher than that in soil inoculated with
either Nephrocytium sp. or with both the green
alga and T. tenuis. The green alga thus apparently prevented development of the nitrogenfixing blue-green alga.
The interaction between green algae and the
nitrogen-fixing algae is further illustrated by the
acetylene reduction activity of the soil. Indigenous nitrogen fixers and T. tenuis fluorished in
the Lina soil, but the activity of both indigenous
nitrogen-fixing algae and T. tenuis was reduced
in the presence of the green alga. Acetylene

918

APPL. ENVIRON. MICROBIOL.

WILSON, GREENE, AND ALEXANDER

TABLE 1. Effect of Nephrocytium sp. on the development of the nitrogen-fixing alga Tolypothrix tenuis in
flooded Lima soil
Chlorophyll
C2H4 formed (nmol/h per cm2 of soil)"
(|hg/CM2of Soil),
Inoculum
Day Day Day Day
7
2
1
10I

Day 1

Day 2

Day 7

Day 10

0.29 - 3.3 1.3 0.055 (0.051-0.065) 0.085 (0.034-0.22) 0.88 (0.38-2.1)

5.5
None
<0.05
0.38 (0.28-0.52) 0.67
Nephrocytium sp.' 5.7 - 4.6 9.1 <0.05
0.39 - 5.8 16
0.32 (0.22-0.48)
3.5
13
(0.99-12)
T. tenuis'
(0.28-580) 35
0.54 (0.12-2.4)
2.2 (0.26-19)
2.1
Nephrocytium sp.' 4.6 - 4.7 5.1 0.20 (0.18-0.21)
and T. tenuisd
"Least significant difference at the 95% level: increase of 2.9 ,tg over that initially present per cm2 of soil.
Values in parentheses indicate 95% confidence interval.
'Initial density, 0.70 ,ug of chlorophyll a per cm2 of soil.
d Initial density, 0.020 ,ug of chlorophyll a per cm2 of soil.

reduction in the dark was not detected, indicating that the algae were the dominant nitrogenfixing organisms in the soils tested. An experimental system was developed to determine
whether communities of algae indigenous to rice
fields could suppress nitrogen-fixing algae.
Effect ofindigenous algae. To demonstrate
the influence of indigenous algae on the development of an introduced nitrogen-fixing alga in
soil, the ability of a number of herbicides to
prevent the development of indigenous algae in
soil was first tested. A variant of a nitrogenfixing alga was then developed that tolerated
one of the herbicides, and the growth of the
resistant variant in soil containing herbicide was
finally compared with growth of the parent alga
in herbicide-free soil containing indigenous algae. If the indigenous algae were limiting the
extent of development of the introduced parent
alga, growth of the variant should be stimulated
by the absence of the indigenous algae, that is,
in the herbicide-treated soil.
Aulosira 68 was subjected to the following
mutagenesis procedure, aseptic techniques being
used in all manipulations. Approximately 100 ml
of an early stationary-phase culture was collected by centrifugation at 8,000 x g for 10 min,
and the resulting pellet was suspended in 45 ml
of 0.35% K2HPO4 adjusted to pH 8.0. N-MethylN'-nitro-N-nitrosoguanidine (2.5 mg dissolved in
5.0 ml of the phosphate solution) was then added
to the suspension. After 30 min at room temperature under normal room illumination, the cells
were collected by centrifugation, and the pellet
was washed once in the phosphate solution and
suspended to its original density in a modification of Fogg's medium (20). To allow time for
phenotypic expression of resistance, the cultures
were incubated at 250C for 14 days in herbicidefree medium without shaking under 50 to 100
microEinsteins per s per m2 or light, and then
5.0 ml of the culture was added to 50 ml of
Fogg's medium containing 1.0 ,ug of simetryne

(3.9-7.7)
(0.42-1.1)
(14-88)
(0.18-24)

per ml. After 17 days in the medium containing

herbicide, 1.0 ml of the culture was transferred
to 50 ml of fresh, herbicide-containing medium.
The culture reached a high density in the second
transfer. This variant was designated Aulosira
68Syl. By using a most-probable-number technique to establish the frequency of events producing resistant variants, it was estimated that
about one such event occurred per milligram of
protein exposed to the mutagen.
The tolerance of Aulosira 68Syl and the parent alga to simetryne was determined by comparing the effect of the herbicide on their growth
rates. The algae were inoculated at an initial
density of 6.4 jig of protein per ml into Fogg's
medium supplemented with simetryne. The cultures were sampled after 0, 24, 48, 72, and 96 h
of incubation at 250C under 50 to 100 microEinsteins per s per m2 of fluorescent light. The
protein and chlorophyll content of the cultures
were determined in duplicate. Growth of the
parent alga, Aulosira 68, was inhibited by 0.2
ug of simetryne per ml and was totally prevented
by 0.8 ,ug/ml (Table 2). In fact, chlorophyll originally present in the inoculum bleached out,
resulting in negative growth rates when the rates
were calculated from increases in chlorophyll. In
contrast, the variant grew at concentrations up
to 0.8 ,ug of simetryne per ml.
The tolerance of the variant to the herbicide
in soil was demonstrated by measuring the instantaneous rate of increase in acetylene reduction activity. All containers received 75 g of
moist soil from an artificial marsh near Ithaca,
N.Y. (50 g of oven-dried soil), 200 ml of distilled
water, and KH2PO4 (20 ,ug of P per ml). Certain
containers received an inoculum of Aulosira 68
or Aulosira 68Syl containing 0.30 mg of protein.
Some containers also received 0.4 or 0.8 ,ug of
simetryne per ml of floodwater. Acetylene reduction was assayed after 5, 10, 15, and 20 days
of incubation.
Aulosira 68Syl but not Aulosira 68 tolerated

NITROGEN FIXATION BY BLUE-GREEN ALGAE

VOL. 38, 1979

0.8 jig of simetryne per ml (Table 2). Acetylene

reduction activity was not detected in uninoculated soil containing 0.8 ,ug of simetryne per ml
of floodwater, indicating that the indigenous algae were also suppressed by this concentration
of herbicide. Growth of the resistant variant in
soil containing up to 0.8 ,ug of simetryne per ml
of floodwater was at least as rapid as growth of
either the variant or parent strain in culture
medium without the herbicide.
To assess the interaction between an introduced alga and the algae indigenous to soil that
had been cultivated to lowland rice, the containers received either 50 g of Crowley soil or 75
g of Moreland soil, equivalent to 42 or 58 g of
oven-dried soil, respectively, as well as KH2PO4
(100 ,ug of P per ml of floodwater). The flooded
soils were inoculated with Aulosira 68 (0.22 mg
TABLE 2. Tolerance of Aulosira 68 and a herbicideresistant variant to simetryne in culture medium
and in soil
Instantaneous increase per day
Inoculum

Simetryne Protein Chloroture

C2H4

culture

0.46
0.49 0.43 + 0.09
0.0
0.16
0.2
0.12
0.28 0.12
0.12 -0.04
0.4
<0.11
0.8
0.01 -0.17
0.49
0.0
0.46
Aulosira
0.46
0.2
0.35
68Syl
0.4
0.30
0.38 0.40 0.16
0.31 0.59 0.21
0.25
0.8
0.90 0.23
0.0
None (in0.26 0.08
0.4
digenous
<0.11
0.8
algae)
a
Instantaneous rates of increase of acetylene reduc-

Aulosira 68

tion activity and their 95% confidence intervals. The

rates were calculated from data collected after 5 and
10 days of incubation in soil without simetryne; after
5, 10, and 15 days in soil with 0.8 ,ug of simetryne per
ml and Aulosira 68Syl; and after 5, 10, 15, and 20 days
of incubation in other soils.

919

of protein in the inoculum) or Aulosira 68Syl

(0.26 mg of protein). If simetryne was not added
to Moreland soil, a bloom of algae developed at
the air-water interface within 2 days. Within 5
days, these algae turned brown and disappeared.
The algae were morphologically similar to the
strain of Nephrocytium isolated from this soil.
Although the density of this bloom was not
determined, visual inspection indicated that the
density of green algae in the Moreland soil was
approximately an order of magnitude less than
the density of Nephrocytium sp. inoculated into
Lima soil in the earlier experiment.
The herbicide at a concentration of 0.8 ,Lg/ml
of floodwater controlled the indigenous algae of
Moreland soil for at least 10 days and in Crowley
soil for 15 days (Table 3). In the simetryneamended soil, acetylene reduction activity was
low, and algae were not visible in the water or
on the soil. Indigenous algae in soils without
herbicide reached near-maximum acetylene reduction activity after 10 days. In Crowley soil,
the suppression of indigenous algae by the herbicide allowed for a greater development of the
introduced alga than was evident in the inoculated but simetryne-free soil, and the activity of
the resistant variant at 10 days was about twice
that of either the parent alga plus indigenous
algae or the indigenous algae alone. In Moreland
soil, however, the indigenous algae did not reduce the extent of development of the introduced alga, and the activity of both the variant
and parent alga at 10 days was about twice the
maximum activity of the indigenous algae.
DISCUSSION
Although a dense bloom of Nephrocytium sp.
profoundly suppressed nitrogen-fixing algae in
Lina soil, the algae indigenous to Crowley soil
had a limited effect on an introduced nitrogenfixing alga, and the species indigenous to Moreland soil had no effect. Perhaps this difference
can be attributed to the initial density of algae

TABLE 3. Effect of indigenous algae on the development of an introduced nitrogen-fixing alga in two rice
soils
C2H2 reduction activity (nmol of C2Hd/h per cm2 of soil)a
Addition

Soil

Crowley

Moreland

None

Aulosira 68
Simetryne
Aulosira 68Syl and simetryne
None
Aulosira 68

Day 5
0.36 (0.17-0.78)

27 (4.0-180)
<0.08
0.37 (0.04-3.2)
<0.08
9.1 (2.1-41)

<0.08
Simetryne
0.35 (0.18-0.67)
Aulosira 68Syl and simetryne
Values in parentheses represent 95% confidence interval.

Day 10

(14-61)
32 (28-37)
0.20 (0.08-0.50)
68 (39-115)
13 (9.2-19)
32 (27-39)
0.36 (0.18-0.75)
33 (22-50)

30

Day 15
12 (9.6-16)
21 (14-30)
0.23 (0.16-0.33)
3.2 (0.14-71)
15 (9.2-24)
14 (5.1-37)
7.3 (0.82-65)
6.1 (0.62-61)

920

WILSON, GREENE, AND ALEXANDER

APPL. ENVIRON. MICROBIOL.

in these soils. The Moreland and Crowley soils

did not contain dense, well-established algal
communities at the beginning of the experiment,
the indigenous algae developing concomitantly
with the introduced species. In contrast, the
nitrogen-fixing alga in Lima soil had to contend
from the beginning with a fully developed bloom
of green algae.
The inhibition of nitrogen-fixing algae by
green algae in Lima soil is consistent with the
observation of Hirano et al. (8) that T. tenuis
could not colonize rice fields unless indigenous
algae were suppressed with CuS04. Similarly,
treatment of rice soils with slaked lime, which
kills indigenous algae, allowed T. tenuis to colonize the floodwaters readily (16). Hence, interactions among algae can be a barrier to the
successful establishment of an introduced nitrogen-fixing inoculum.
The potentially inhibitory interactions among
algae in soil include competition and amensalism. In natural waters, it has been occasionally
possible to identify which interaction is operating. Titman et al. (14) showed that competition
allowed the stable coexistence of two diatoms;
when the ratio of concentrations of phosphate
and silicate was in a certain range, the demand
of one alga for phosphate produced a phosphate
concentration that was growth limiting for the
second alga, and the demand of the second alga
for silicate produced a concentration that was
growth limiting for the first alga. Similarly, Klotz
et al. (10) showed that Chlorella sp. excluded
Achnanthes deflexa from river water by competition. On the other hand, Keating (9) attributed the dominance of blue-green algae in a
eutrophic lake to its release of a heat-labile
substance toxic to green algae and diatoms, and
Murphy et al. (11) showed that iron-chelating
compounds excreted by blue-green algae were
toxic to other algae.
The type of interaction that may occur among
algae has important agricultural implications because competition can be relieved by fertilizing
with the limiting nutrient, but amensalism can
only be overcome by suppressing the organisms
that are responsible. The soil used in this study
was amended with sufficient phosphorus to insure that the element was not limiting, and
nitrogen would not be limiting for nitrogen-fixing
organisms. If competition for a nutrient limited
the nitrogen-fuing algae, the substance in short
supply was probably carbon, sulfur, or a trace
element. De and Sulaiman (7) reported that C02
availability limited nitrogen fixation by algae in
two rice soils from India, but sulfate was not
limiting in the soils they studied. However, a
competition for nutrients probably does not ex-

plain the suppression of the nitrogen-fixing algae

by the dense blooms of Nephrocytium sp. There
was little growth of Nephrocytium sp. after day
1, so this alga probably did not act as an ongoing
sink for nutrients, yet development of indigenous, nitrogen-fixing algae and T. tenuis was
inhibited for at least 10 days, suggesting that
amensalism may have been a factor.
Use of herbicide-resistant algae is a useful tool
for the detection of interactions among algae in
complex model ecosystems. The methodology
developed in the laboratory-scale experimental
model should be easily adaptable to field studies,
allowing experimental manipulation of populations of algae in ways that were previously impossible. Herbicide-resistant algae may also be
agriculturally useful. If a herbicide used for weed
control in rice fields also kills algae, inoculation
with a resistant alga should restore an important
source of biologically fixed nitrogen. If rice fields
routinely develop dense blooms of green algae,
use of the herbicide and a resistant alga may
even stimulate nitrogen fixation by these organisms.
ACKNOWLEDGMENTS
We thank W. Bollick for the Louisiana soils.
This research was supported in part by a grant from the
United Nations Development Program.
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