HIGH
NITROGEN
FIXATION
SEA AND THE
RATES IN THE
ARABIAN
SEA1
SARGASSO
Richard C. Dugdale and John J. Goering
Institute
of Marine
Science, University
of Alaska, College
and
John H. Ryther
Woods Hole Oceanographic
Institution,
Woods
Hole, Massachusetts
ABSTRACT
Nitrogen fixation rates have been mcasurcd in the Sargasso Sea and the Arabian Sea
using the N'" method. Heavy enrichments in N1" wcrc found in a number of experiments in
material.
which the blue-green
alga, Trichodemnium, was used for the experimental
INTRODUCTION
In a previous communication
(Dugdale,
Menzel, and Ryther 1961)) we reported
the results of an experiment in which low
nitrogen fixation rates associated with the
blue-green
alga, Trichoclmnzium,
were
measured using the N15 method. We have
recently obtained confirmation
of largescale nitrogen fixation in the Sargasso Sea,
where the original experiment was carried
out, and in the Arabian Sea.
METHODS
The N15 method for detecting nitrogen
fixation has been adapted by Ncess et al.
(1962)
to the measurement of fixation
rates in aquatic communities. The following procedure is carried out after placing
water containing the desired organisms in
a l-liter flask having a standard taper joint
at the neck and into which a specially designed gas flushing unit is fitted: 1) Atmospheric Na ( as well as other gases) is
flushed from the water with a mixture of
80% He and 20% 02 at a pressure of 0.8
atm; 2) 0.2 atm Nz enriched to 95% N1’) is
added to the flask and equilibrated
with
1 Contribution
No. 3 from the Institute of Marine Science and No. 1510, from the Woods Hole
Oceanographic
Institution.
This rcscarch was supported
by National
Science Foundation
Grant
CB-24 and by the United States Biological
Program of the International
Indian Ocean Expedition.
We wish to thank Dr. F. A. Richards, Department
of Oceanography,
University
of Washington,
for
the Consolidated-Nicr
mass spectrometer
used in
thcsc studies.
the aqueous phase by shaking; 3) the
sample is incubated in the flask under the
conditions selected for the experiment; 4)
the particulate fraction is captured on a
glass filter (Hurlburt 984H) and converted
to molecular nitrogen by a Dumas combustion ( Dugdalc and Barsdate 1964); 5) the
NE/N14
ratio of the resulting N2 is dctermined with a mass spectrometer, the
ratio converted to atom per cent NLB, and
the enrichment over the normal atom per
cent Nz” of the organic material calculated.
Isotope ratios were measured with either
a Consolidated-Nier
21-201 magnetic mass
spectrometer or with a Bendix 17-210 timeof-flight mass spectrometer. The atom per
cent N’S values in Table 2 that have two
significant figures to the right of the decimal point were obtained with the time-offlight spectrometer.
All other measurcmcnts were macle with the magnetic spectrometcr. The atom per cent excess IV5
associated with each flask has been calculated by subtracting the control (normal
atom per cent N1” of Trichodesmium)
or
by subtracting
the average of the five
Bermuda controls (0.349) given in Table 1.
RESULTS
Results for the Sargasso Sea nitrogen
fixation cxpcriments conducted at Bermuda
are given in Table 1. Material for these
experiments was collected about 4 km from
Rermuda by towing a No. 8 or No. 20
plankton net at the surface. Upon return
to the laboratory, individual
colonies of
507
508
RICHARD
C. DUGDALE,
TABLE
__---.-_- ~_
.----______
__ --
JOHN
J. GOERING,
AND
Sargasso Sea fixation
1.
.-
-
-
----
~._
JOHN
II.
RYTIIER
results
=
.-
Length
incubation
-of
(hr)
--
-.
Atom %
Nl5 excess
Date
-.-.
Snmplc
__ ~
4 Sept 1962
Control
Flask 1
Control
Flask 1
Flask 2
Control
Flask 1
Flask 2
Flask 1
Flask 2
Control
Flask 1
Flask 2
Flask 3
Control
Artificial
light
Artificial
light
Artificial
light
Direct sunlight
Direct sunlight
Dark
Artificial
light
56% of incident light
80% of incident light
100% of incident light
-
13
18
24
8
8
24
24
4.5
4.5
4.5
-
0.348
0.350
0.348
0.349
0.356
0.350
0.518
0.566
0.353
0.358
0.350
0.399
0.377
0.384
0.351
Flask
5 hr direct sunlight,
15 hr artificial light
20
0.559
0.208
5 hr direct sunlight,
15 hr artificial light
20
0.447
0.096
-.
11 Scpt 1962
20 Sept 1962
1 Ott
1962
12 Ott
1962
19 Ott
1962
1
Flask
-.--
--
_-----.
~
-~
2
.-
~-
Light
-.-
----
Trichodesmium
were separated from the
other plankton and placed in fixation flasks
containing
Millipore@
( 0.45 p) -fil tercd
surface seawater. After treatment with
N215 the flasks were incubated for various
lengihs of time either in artificial
light
(16,000 lux) at 2OC, or in a seawater-cooled
box exposed to direct sunlight. The box was
fitted with neutral density filters for the 12
October 1962 experiment.
In some experiments
(20 September
1962, 19 October 1962), the rate of fixation
was about 20 times the highest rate reported in the earlier communication (Dugdale et al. 1961). In the remainder, fixation
was low or undetectable. The, highest rates
observed here are comparable to fixation
rates observed in lakes during periods of
nitrogen-fixing
blooms ( Dugdale and Dugdale 1962). The 19 October 1962 experiment, in which 1 pug-at. NH,+-N/liter
was
added to flask 2, suggests that NHh+-N
inhibits fixation. That a certain degree of
variability
occurs within a given experiment may be seen in the 20 September 1962
results, in which like treated flasks showed
~~
2 Rcgistercd trademark,
Millipore
ration, Bedford,
Massachusetts.
Filter
Corpo-
--
^%
%
0.002
0.001
0.008
0.169
0.217
0.004
0.009
0.049
0.027
0.034
-
a difference in rate of fixation. Some of the
experimental
conditions that may affect
fixation will be discussed later.
The results of nitrogen fixation measuremcnts made during Cruise 4A of the U.S.
Biological Ship for the International Indian
Ocean Expedition, the RV Anton Bruun,
arc summarized in Table 2. The majority
of the stations is located in the northern
Arabian Sea; Stations 174 and 176 lie farther south along the coast of Saudi Arabia.
The enrichments in Nr5 observed are similar to those shown in Table 1, the value for
the 4-hr experiment on Station 188, 4.65
atom per cent excess, being the highest
measured in the sea to date.
Colonies of Trichodesmium
were used
for experimental material in all the experiments except at Station 183, where a large
quantity of Rhizosoknin was collected from
the surface, and at Station 194, where a
heavy bloom of Noctiluca occurred. Two
distinct forms of Trichodesmium were observed in the No. 20 mesh nets normally
towed for 10 min or less at 1, 10, 20, 30,
and 40 m, one occurring as green bundles
of filaments and the other as larger brown
spherical clumps of filaments corresponding to the form wc have worked with in the
NITROGEN
FIXATION
TABLE
D:1tc
17
18
28
29
31
1
1
1
3
3
Ott
Ott
Ott
Ott
Ott
Nov
Nov
Nov
Nov
Nov
Station
1963
1963
1963
1963
1963
1963
1963
1963
1963
1963
174
176
183
184
188
190
Flask a
Flask h
191
192
194
195
IN TI-LE SARGASSO
2.
Inclian
Latitude
AND
ARABIAN
Ocean fixation
rmults
Longituclc
16”27’ N
54”40’
57”09’
16”28’ N
23”42’ N
66”21’
22”34’ N
65”50’
23”21’ N
64”52’
61”39’
24”47’ N
10 colonies
Numerous colonic :s
23”57’ N
60”58
60”36’
23”08’ N
60”06’
22”22’ N
21”32’ N
60”40
Sargasso Sea. At six stations, the former
type was observed at virtually
all the
depths mentioned above; the latter appeared and became abundant in the collections at Stations 191, 192, 193, and 195,
all in northwestern Arabian Sea near the
Gulf of Oman.
When the negative results with RhixosoBenin (Station 183) and No&&a
(Station 194) are removed from consideration,
it becomes clear that high enrichments
occurred in a large proportion
of the
Trichodesmium
experiments, that is, in
five out of eight possibilities.
Cloudiness,
suggesting the presence of bacteria, devcloped quickly in the incubation flasks from
Stations 191 and 195, a possible explanation
for the failure to observe nitrogen fixation
at these stations, which showed heavy concentrations of the brown form of Trichodesmium. Strong fixation occurred in expcrimcnts using the brown form exclusively
(Station 192) and in those using the “green
bundles” form ( Station 190).
DISCUSSION
The variability
obscrvcd in these measurcments (see below) precludes certain
levels of speculation regarding the significance of these data. However, we consider
it a virtual certainty that the large-scale
blooms of Trichodesmium
reported from
tropical oceanic regions are indeed nitrogen-fixing
blooms analogous to those
observed in lakes associated with scvcral
species of Anabaena by Dugdale and Dug-
E
E
E
E
E
E
E
E
E
E
5Q9
SEAS
Length of
incubntion (hr)
Atom %
N’”
Atom %
NlG cxccss
32
5.5
?
4
0.719
0.387
0.366
1.024
5.00
0.370
0.038
0.017
0.675
4.65
5
5
10
30
28
38
0.81
0.47
0.352
0.902
0.354
0.354
0.46
0.12
0.003
0.553
0.005
0.005
dale ( 1962) and by Goering ( 1962).
Certain reservations must be made; for
example, our experiments do not prove that
Trichodesmium is itself able to fix nitrogen.
However, this is unimportant
from the
point of view of the ecologist, since the
ability to fix nitrogen has been clearly
shown to lie with the Trichodesmium colonies (that is, the alga and any associated
bacteria or fungi ) .
Up to this point we have not been concerncd with the species composition of the
experimental material beyond noting obvious macroscopic differences. Three species, T. eythraeum Ehrcnb., T. hildebrwtii
Gom., and T. thiebautii Gom., are reported
for the Indian
Ocean by Desikachary
( 1959), and McLeod, Curby, and Bobblis
(1962) suggested that two or more species
may have been present in their collections
at Bermuda.
The data obtained so far have been
characterized by a disturbing lack of consistency, that is, experiments at Bermuda
separated by only a few days give highly
divergent results, and the same is true for
the cruise data. WC suspect that a large
portion of this discrepancy may lie in the
experimental method. Now that we have
obtained N1” enrichments of a high order,
replication and suitable experimental design should yield insight into the problem.
McLeod et al. (19612) report a twentyfold
variation in the rate of photosynthesis from
cells of a given collection of Trichodesmium.
In some collections, cells were present that
.
510
RICHARD
C. DUGDALE,
JOHN
J. GOERING,
would not photosynthesize, and other cells
would do so only after a period of adaptation
to light. Therefore, some of the variability
from day to day and within any single experiment may be the result of differences
in the physiological condition of the colonies. Menzel (1962) has also shown an
autoinhibition
of photosynthesis by Trichodesmium at Bermuda; at Station 190 flask
n contained only 10 colonies and fixed
nitrogen at double the rate of flask b, that
contained numerous colonies. The organisms may also be sensitive to other features of the technique such as the length
of sparging and composition of the sparging gas.
REFERENCES
DESIKACHARY, T. V. 1959. Cyanophyta.
Academic, New York. 686 p.
DUGIIALE, R. C., AND R. J. BARSDATE. 1964.
Rapid conversion
of organic nitrogen to Ns
AND
JOHN
H. RYTIIER
for mass spcctrometry
by an automated
Dumas procedure. ( Unpublished
manuscript. )
-,
D. W. MENZEL, AND J. II. RYTHER.
1961. Nitrogen fixation in the Sargasso Sea.
Deep-Sea Rcs., 7: 298-300.
DUGDALE, V. A., AND R. C. DUGDALE. 1962.
Nitrogen
metabolism
in lakes II.
Role of
nitrogen fixation in Sanctuary Lake, Pennsylvania.
Limnol. Occanog., 7: 170-177.
GOEIUNG, J. J. 1962. Studies of nitrogen fixation in natural fresh waters.
Ph.D. Thesis.
Univ. of Wisconsin.
133 p.
McL~on,
G. C., W. A. CURBY, AND F. BOBBLIS.
1962. The study of the physiological
characteristics of Trichodesmium
thiebnutii.
A.E.C.
Rcpt. Contrib.
AT( 30-l ) 2646.
Bermuda
Biological
Station.
13 p.
MENZEL, D. W.
1962. Inhibition
of photosynthesis by Trichodesmium
in the Sargasso Sea.
A.E.C. Rcpt. Contrib. AT(30-1)
2646. Bcrmuda Biological
Station.
6 p.
NEESS, J. C., R. C. DUGDALE, V. A. DUGIJALE, ANU
J. J. GOERING. 1962. Nitrogen
metabolism
in lakes. I. Measurcmcnt
of nitrogen fixation with N”.
Limnol. Occanog., 7: 163-109.