NITROGEN FIXATION ASSOCIATED WITH THE MARINE MACROALGA,
CODIUM FRAGILE
A thesis submitted to the faculty of
San Francisco State University
in partial fulfi I lment of the
requirements for the
Master of Arts
degree
by
WILLI AM D. HEAD
San Francisco, California
ACKNOWLEDGMENTS
I would I ike to dedicate this work to Mary Silver.
Her friendship 1
patience, and vitality provided me with encouragement when I was first
floundering as a graduate student.
I am deeply indebted to Dr. Ed Carpenter for sponsoring me during
my fellowship at Woods Hole Oceanographic Institute and for introducing
me to the problem of N2 fixation. It was through his lab that the bulk
of this research was completed and I thank him for his advice and friendship.
I would also I ike to express my gratitude to J. Sayles, C. Price,
J. P. Clarner, B. W. Schroeder, N. Corwin, V. Peters, and K. Ulmer for
their assistance; H. Jannasch, J. Goldman, and A. Col I ins for their
helpful discussions; and M. Mandel for determining the bacterial guanine/
cytosine ratios.
Appreciation is given to Drs. John Martin and Hideo Yonenaka for
serving on my committee and offering critical suggestions during the
preparation of this manuscript, and to Moss Landing Marine Labs for
introducing me to the ocean world.
Last, but not least,
wish to
acknowledge inspiration from John Oliver's insanity.
This work was supported by National Science Foundation Grant
GA 37993 and a W.H.O.I. summer student fellowship.
iii
TABLE OF CONTENTS
ACKNOWLEDGMENTS.
ABSTRACT ••
INTRODUCTION
•
•
..... .
•
•
•
•
•
•
Page
•
•
•
•
•
•
0
•
•
•
•
•
•
•
•
•
•
•
iii
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
0
v
..........................
METHODS AND MATERIALS.
0
•
•
•
•
0
•
0
•
•
•
•
•
•
•
•
•
•
•
•
•
3
RESULTS ••
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
0
•
•
•
8
•
•
•
•
•
0
•
•
•
•
•
•
•
•
•
•
•
•
0
•
•
•
8
•
Light Effects •
...........
N2 Fixation in a Shallow Coastal Bay. . . . . . . . . . . .
Agent Responsible for N2 Fixation •• . . . . . . . . . . .
Antibiotic Treatment. . . . . . . . . . . . . . . . . . . .
DISCUSSION . . . . . . . . .
LITERATURE CITED
Availability of Combined-N Compounds.
•
•
•
•
•
•
8
12
16
19
•
•
•
•
•
0
•
•
•
•
•
•
•
•
•
•
•
•
21
•
•
•
•
0
•
•
•
•
•
•
•
•
•
•
•
•
•
28
iv
ABSTRACT
Nitrogen fixation at rates of up to 7.3
~g
N fixed g dry wt-1 hr -1
2
is associated with the marine macroalga, Codium fragile.
A bacterium
identified as an Azotobacter has been isolated from Codium that is
capable of fixing atmospheric nitrogen in pure culture under aerobic
conditions.
Scanning electron micrographs showed dense populations
of rod-shaped bacteria, presumed to be Azotobacter, on the surface
of Codium.
When Codium photosynthesis is decreased by I ight shading, N
2
fixation drops proportionally, thus suggesting that N fixation is
2
coupled to the release of dissolved compounds by Codium. Addition
of glucose to seawater increases N2 fixation indicating that the agent
of N fixation is a heterotroph.
2
In a field survey, an inverse relationship was noted between N
2
fixation and the concentration of combined-N compounds in seawater.
At total available N (N0 - , N0- , NH+, urea) concentrations greater than
2
4
3
-I
10 ~g-atom I iter , no N fixation was associated with Codium. At
2
Nobska Beach the total N-input through N fixation in the summer was
2
2
2.0 mg m- day-l for water columns ranging between I and 3 m, and 0.47
mg m- 2 day-l in the 3 to 5 m stratum.
The importance of N fixation to
2
the nitrogen budget of Codium and shallow bays is discussed.
v
INTRODUCTION
Very little is known about the relationship between macroalgae
and attached N fixing bacteria.
2
Reinke (1903) suggested that, since
Azotobacter occurs on the surface of some marine algae, a symbiosis
may exist in which the algae supply the bacteria with carbohydrates
as a source of energy and utilize the nitrogen fixed by the bacteria.
lssatchenko (·1926) demonstrated the presence of Azotobacter and
Clostridium strains on Fucus, and Pshenin (1963) noted a rich population
of Azotobacter on the surface of Phyl lophora.
However, it was not
determined in these investigations whether the Azotobacter fixed nitrogen
~situ,
and Reinke's (1903) hypothesis is not yet verified.
Stewart (1971) found no evidence
of~
Recently,
situ N fixation on the larger
2
marine algae of Scotland and suggested that if nitrogen fixation by
heterotrophic bacteria did occur, it was probably unimportant.
S. W. Watson and R. Nairn (personal communication) have determined
that N fixation is associated with the marine chlorophyte, Codium
2
fragile. Codium may be described as a rapidly colonizing invader
that has affected the ecology of the New England coastal zone.
Codium
was first documented on the Atlantic coastline on Long Island Sound
by Bouck and Morgan (1957).
Since then it has established itself
in coastal waters from Maine to New Jersey and it is sti I I undergoing
expansion of its geographic range (Malinowski and Ramus, 1973).
attaches to a variety of substrates.
Codium
According to Ben-Avraham (1971),
gravel beaches may evolve through the attachment of Codium to smal I
rocks that may subsequently be transported ashore during storms.
2
Similarly, the attachment of Codium to I iving oysters and scallops
has damaged the commercial shel If ish harvest in some areas of New
England (Ramus, 1971).
The accumulation of masses of dead Codium
on beaches is also aesthetically unpleasant.
This study is an investi-
gation of the organism responsible for, and factors affecting Codium's
reported N fixation. Additionally, data is presented on the input
2
of nitrogen to a coastal bay by the N fixation associated with Codium.
2
3
METHODS AND MATERIALS
Whole plants of Codium (ca 20~25 em long) were collected off
0
0
Nobska Point, Woods Hole, Massachusetts (41 31' N; 70 40' W) and
placed into 3-1 iter Fernbach flasks containing filtered seawater.
replicate flasks were incubated in sun I ight for three hours
Two
un~er
neutral-density screens (plastic window screening) corresponding to
the 100, 60, 30, 15, 3 and O% I ight level and cooled with running sea
water (20° to 24°C).
Oxygen production was measured by the Winkler
method (three replicates for each flask) and carbon uptake was estimated
from oxygen production by assuming a photosynthetic quotient of 1.2 (Strickland and Parsons, 1968).
The plants were rinsed in distil led water
after each experiment and dried to a constant weight at 60°C.
Codium was also assayed for associated N fixation by the acetylene
2
reduction method (Stewart, 1971). The technique was modified to
accommodate large algae.
Segments of Codium (ca 5-6 em long) taken
from the portion of the thai Ius near the dichotomies were placed in
50 ml erlenmeyer flasks containing 25 ml filtered seawater and capped
with serum stoppers.
to prevent leakage.
The stoppers were sealed with silicone grease
Five ml of high-purity acetylene gas were injected
Into the bottle with a gas-tight syringe and the excess pressure released
by piercing the serum stopper with a hypodermic needle.
Control flasks
contained filtered seawater and Codium samples fixed with 50 per cent
trichloroacetic acid (TCA).
Two replicate samples were incubated in the
manner described for primary production.
After two to three hours, the
4
gas phase was ana I yzed on a Packard (mode I 417) gas chromatograph
0
fitted with a Porapak R column (2.7 m by 0.3 em) at 50 C.
as a carrier gas with a flow rate of 40 ml min-I.
N served
2
The·rate of
acetylene reduction was converted to N reduction by assuming a molar
2
ratio of 3: I (Scholl horn and Burris, 1967; Hardy et ~._, 1968).
Samples of Codium were also analyzed for eel lular carbon and nitrogen
concentrations with a Perkin-Elmer (model 260) elemental analyzer.
Codium was cultured for 14 days in nitrogen-enriched and nitrogendeficient media to assess the effect of inorganic nitrogen on N fixation
2
rates. ·Three, 3-liter Fernbach flasks containing Sargasso Sea water
lacking measurable No;, No;, or NH+
4 were treated in the following manner:
One flask was enriched with complete f/2 culture medium (Gui I lard and
Ryther, 1962) with No; (883 ~M NaN0 ) as the nitrogen source; another
3
flask was enriched with f/2 with NH: (616 ~M NH CI) as the nitrogen
4
source; and the third flask was enriched with f/2 lacking inorganic
nitrogen.
Entire plants were placed into the flasks and incubated in
a walk-in incubator at 16° C under 4,000 lux from cool-white fluorescent
I ight with a 16 hr I ight and 5 hr dark cycle.
The water was constantly
stirred with a magnetic stirring bar and aerated using air filtered
through activated charcoal and passed through a IN HCL solution to
remove atmospheric NH+ • Segments of Codium were removed from each flask
4
every two days and assayed for N fixation. Three replicates were run
2
for each treatment.
A field survey was carried out to determine the relationship
between the concentration of combined-N compounds in seawater and the
rate of N2 fixation associated with Codium.
Codium was collected at
5
various sites along the shore of Buzzard's Bay, Cape Cod, Martha's
Vineyard, and Nantucket and the rate of N fixation was determined
2
after incubating for two hours in an i I luminated incubator box at 20°C
under 3,500 'lux from cool-white fluorescent I ight.
Seawater samples
...
+
were also collected and frozen for analysis of No;, NOy NH (Strickland
4
and Parsons, 1968) and urea (Newell et ~., 1967). At each collection
site the abundance of Codium was estimated by SCUBA or skin diving and
recorded as either absent, present in moderate concentrations (ca <one
-2
-2
plant m ), or high density (ca >one plant m ).
At one site, Nobska
Beach on Vineyard Sound; Codium standing crop measurements were made.
This was done by randomly throwing a I m diameter metal ring and harvesting all of the Codium within the ring.
between
Two depth intervals were surveyed,
and 3m and 3 and 5 m, and seven measurements of standing
crop were made with the quadrat in each depth interval.
Codium wet
and dry weights were then determined in the laboratory.
Glucose enrichment experiments were performed to determine if N2
fixation was stimulated by additions of a carbohydrate source.
Whole
plants of Codium were placed into 3-1 iter Fernbach flasks containing
glucose solutions of 0.01, 0.1, 1.0, and 10.0 g glucose· liter
-I
and
incubated in a walk-in incubator at 16°C under 4,000 lux from coolwhite fluorescent light for one hour.
Segments were then.transferred,
along with 25 ml of the solution, to 50 ml erlenmeyer flasks and assayed
for N fixation after a two hour incubation period.
2
Two replicates were
run for each treatment.
N fixing bacteria were isolated
2
fro~
Codium by first aseptically
passing the alga through several washings of sterile seawater and then
6
either rubbing
th~
Codium·across agar plates or scraping the C6dium
with an inoculating loop and streaking the loop onto agar plates.
The
culture medium contained O.J .mg K HP0 , 0.5 mg NaMo0 "2H o, 0.005 mg
4 2
4
2
CaS0 "2H o~ 0.2 mg MgS0 "7H 07 0.3 mg ferric citrate, 10 g sucrose,
4 2
4 2
0.05 mg yeast extract, 15 g agar, 1000 ml filtered seawater.
The
bacteria were incubated at room temperature (ca 25°C) and individual
colonies were selected for axenic culturing.
An isolate was found
to fix N and it was maintained on an artificial seawater medium
2
containing 25 g NaCI, 8 g MgS0 "7H 0~ 5 mg NaH Po "H 0, 3.5 mg FeCI "
4 2
2 4 2
3
6H 2o, 0.01 mg CuS0 4 "5H 0, 0.02 mg ZnS0 "7H 0, 0.1 mg CoCt "6H o, 0.18
2
2 2
4 2
mg MnCt 2 •4H 2o, 0.5 mg Na Mo0 "2H 2o, 10 g sucrose, 15 g agar,. 1000 ml
2
4
disti I led water; pH adjusted to 7.5 with NaHC0 •
3
Samples of C6dium were prepared for scanning electron microscopy
of the bacteria on the surface of the alga.
Smal I sections of C6dium
were fixed in 2% osmium tetroxide, washed in dilutions of ETOH and freon,
and dried in a critical-point dryer.
Samples were plated with gold
just before observation.
Codium was treated with antibiotics in order to kil I or inhibit
the N fixing organisms on the alga's surface. 200 mg of penici I I in G
2
and 100 mg of streptomycin sulfate were dissolved in 5 ml of distil led
water and sterile filtered through a 0.22
~m
membrane fi Iter apparatus.
The solution was aseptically transferred to 2 I iters of autoclaved
seawater.
Whole plants of C6dium were placed in this solution and a
solution lackiRg antibiotics and incubated in a walk-in incubator as
described previously.
Three replicates were run for each
treatment~
Complete inhibition of N2 fixation on C6dium treated with antibiotics
7
was observed after 24 hours.
The plants were then washed in sterile
seawater and each plant was divided in half at the basal disc.
One
half of each plant was inoculated with N2 fixing bacteria isolated
fro~
C6dium by
swab~ing
the surface of the alga with an inoculated
loop of the bacteria, while the other half of each plant was not
inoculated with the bacteria.
The plants were then placed in
3~1
iter
Fernbach flasks containing 2 I iters of autoclaved seawater and incubated
in a walk-in incubator as described previously.
Segments of Codium
were removed from each flask every two day and assayed for N2 fixation.
Three rep I icates were run for each treatment.
8
RESULTS
Light Effects:
Codium incubated at different I ight intensities
exhibited a positive relationship between I ight intensity and N2
fixation (Figure 1).
Both N2 fixation and phot9synthesis decreased
with a decrease in I ight intensity.
There was_a positive correlation
(r=.9, p<.001) between net production and N2 fixation (Figure 2).
_l
Maximum net photosynthesis was 2.0 mg C g dry wt
_l
maximum N2 fixed was 0.6
~g
N g dry wt
_l
hr
and the
_l
hr
Nitrogen fixation
did not respond immediately to changes in I ight intensity and it was
necessary to condition the samples at the desired I ight intensity for
at least one hour prior to acetylene injection.
N2 fixation did not
occur when Codium was incubated in the dark.
Avai labi I ity of combined-N compounds:
When maintained in a medium
with no added nitrogen compounds (flask A) the rate of N2 fixation
associated with Codium increased stead! ly with time (Figure 3).
Media
with added N compounds (flasks Band C) showed an initial decrease
in N2 fixation unti I day 4 and then a rise in N2 fixation up to day 14.
Trace amounts (<1.0 ~M) of No; and NH! remained in the seawater from
flasks Band C.
Previous experiments (Head and Carpenter, unpublished
data) indicated that when N03 and NH! were supplied together as a
nitrogen source, Codium removed alI of the NH! before uti I izing N0 3 ~
N2 fixation rates were lowest in the medium containing NH! as the
nitrogen source (flask C).
Primary production measurements conducted
at the end of the experiments indicated that the plants from the flasks
were alI actively photosynthesizing.
Individuals of Codium collected for
9
_J4
1
w
d
A.JP 5 G3Xl.:l lN
JM
1
orr
N
¢
d
d
0
0
\
\
\
\
\
\
\
~
_J4
1
Figure I.
~
C\1
JP•\
1
k.1p 5_
~
o;;;t
0
5w
Net photosynthesis and N fixation as a function of light
2
level. Ranges are Indicated by vertical I ine through mean
datum point (two rep I !cates).
10
0.6
0
I
L.
..c
,_ 0.4
3
0
>..
0
L.
"0
O'l
0
w
X
lL
C\1
z
0.2
0
O'l
:l..
0
Figure 2o
1.0
NET PRODUCTION
(mg C g dry wf1hr- 1)
2.0
Regression of N fixation on net production of Codium
2
tragi le. Pooled data from exp. I (I) and exp. 2 (O)o
II
''-
..c
-
I
3:
>.
'-
"0
0'1
0
1.0
w
X
LL
z
N
0'1
=a
0
4
6
8
DAYS
Figure 3o
Time course of the response of N fixation in media lacking
2
inorganic nitrogen (A); and N fixation in media enriched
2
with NO; (8) and NH; (C)o Ranges are indicated by vertical
I ine through mean datum point (three rep I icates).
14
12
these experiments appeared I ight green in color and were coated with
whitish hairs.
When cultured in a nitrogen rich medium (flasks Band C);
however, Codium lost its hairs and turned dark green while the Codium
in the nitrogen deficient medium (flask A) retained its hairs and became
a I ighter green.
This bleaching effect under low nitrogen concentrations
has been recorded for some red algae (Yamada, 1961; Neisch and Fox, 197a).
.
)
In the field it was evident that ambient combined-N concentrations
affected N2 fixation.
Codium, if present, was collected at each of
the sample locations shown in Figure 4.
Places where Codium was rare
or absent had high wave action or sandy bottoms without suitable
attachment sites.
+
NH~,
Concentrations of combined-N compounds (No;, No;,
urea) in seawater at each site were measured then summed and plotted
against the rate of N2 fixatJon.
A negative correlation (r=-.7, p<.001)
was observed between the two parameters (Figure 5).
Nitrate and nitrite
were present in low (<1.0
~M)
alI but one sample site.
At combined-N concentrations over about 10
~g-at
I iter
_l
or below detectable concentrations at
there was virtually no observable N2 fixation.
Codium
collected from areas of high nitrogen concentrations (e.g. boat harbors)
appeared dark green while Codium collected from areas of low nitrogen
concentrations (e.g. exposed coasts) appeared I ight green and were
covered profusely with whitish hairs.
N2 fixation in a shallow coastal bay:
Based on an average wet to
dry-weight ratio of 12:1 (Figure 6) the standing crop of Codium at Nobska
_2
Beach in July averaged 153±:62 g dry wt m
(95% confidence I imits of
_2
the mean) in the 1 to 3 m depth interval and 65 ± 47 g dry wt m
3 to 5 m interval.
in the
The average rate of N2 fixation between 1 and 3m
13
t----c=:r----r:=:r----r:=::::r--t=::r---;=:=r---;c--,--l--,--,----,-,----,--I-,-I4J 0 00 I
1
7
69°30
Figure 4. Survey conducted along Cape Cod, Buzzard's Bay, Martha's
Vineyard, and Nantucket for relative density of Codium
fragi Ie.
I
in dense beds;
e
scattered; 0 absent.
14
•
• •••
, I
I-
_c
I..._
I-
• ••
O'l
1>;9
3
>.
-o
..Y~
C?o
0
w
lL
c:\.1
O'l
::J .
\5'..>
• •
X
z
••
• •
0.1
'o·/c9
•
0:,.
•
•
•
•
•
0
2
4
•
6
12
COMBINED-N (No;, No;,NH;,urea)
( ug-atom liter- 1)
Figure 5.
N2 fixation as a function of combined-N (No;, No;, NH;, urea)w
15
1500
0
0
...-...
E
.!?:1000
II
(,9
w
$:
1--
w
$: 500
50
100
DRY WEIGHT (gm)
Figure 6.
150
Regression of wet weight of Codium tragi leon dry weight of
c~ tragi le.
16
on cloudless days in July and August was 1.1 ± 0.42
_l
wt
~g
N2 fixed g dry
_l
hr
_l
and between 3 and 5 m was 0.6 ± 0.25
~g
N2 fixed g dry wt
_l
hr
Agent responsible for N2 fixation:
Codium itself could not fix
nitrogen since this process is confined to procaryotes.
Epiphytic
blue-green algae (Myxophyceae) also were not responsible since microscopic examination of Codium showed that most plants used in the
assays were free of these organisms.
Nitrogen fixation associated
with Codium appeared to be heterotrophic, since additions of up to
1.0 g glucose I iter
_l
stimulated N2 fixation (Figure 7 ).
However,
_l
glucose at 10 g I iter
showed no stimulatory effect.· Examination of
the plants with scanning electron microscopy revealed the presence of
numerous attached rod-shaped bacteria on the anastomosed surface of
the alga (Figure 8).
A gram-negative rod-shaped bacterium was isolated
from the Codium collected at Nobska Beach in July 1973.
During the
isolation attempts 41 bacterial isolates were obtained of which 4 were
able to fix N2 •
In pure culture the isolate is rod-shaped during
exponential growth and spherical in older cultures.
These morphological
changes are typical of the genus Azotobacter (Johnstone, 1974).
The
bacterium is capable of fixing nitrogen in pure culture under aerobic
conditions and these properties are also characteristics of this genus.
Laboratory experiments on the bacterial isolate indicated that
it wi I I grow in the previously noted bacterial medium without salts
added.
It is able to uti I ize either glucose, mannitol or sucrose
individually in pure culture as its sole source of energy.
grow in peptone broth with and without added glucose.
It wi I I
Its growth in
17
0
0
d
l
I
I
I
I
I
--o--
0
-
/
/
/
-
/
/
T,_
-
/
(I)
/
/
0'1
..._,.
/
/
---o--
""'\..
w
Ci (J)
0
0
:::)
_j
(!)
........
"'
·'"
''
0
----o----9\
d
\
\
\
~
0
0
q
_l4 1JM
1
Figure 7.
1.0
d
0
,(Jp
5 G3Xi.:i ~N 5rr
N fixation as a function of glucose concentration. Results
2
of two experiments. Ranges are indicated by vertical I ine
through mean datum point (two rep I icates).
18
A
B
Figure B.
Scanning electron micrographs of A) anastomosed surface of
Codiu!Jl~.•
line equals 100 ]lm; B) bacteria on su
of utricles, I ine equals 5 ]lm.
ace
19
_1
media with N0 3 concentrations above 1 g I iter
to improved growth below 1 g 1iter
_1
is I imited as compared
The isolate is an obi igate aerobe
and wi I I not grow when the 0 2 is eliminated from I iquid culture by the
purging with N2.
Also it wi I I not grow when inoculated within agar test
tube slants but wi I I grow at the surface of the medium where 0 2 is
present.
Guanine to cytosine (G/C) base ratio of the isolate is 60.2%. j
AI I of these properties are characteristic of the genus Azotobacter
(Johnstone, 1974).
A culture of this bacterium has been deposrted
with the American Type Culture Collection in Rockvi I le, Maryland.
Antibiotic treatment:
N2 fixing organisms on Codium were inhibited
or killed after treatment with antibiotics and N2 fixation was restored
by inoculating the surface of Codium with the N2 fixing bacterial
isolate (Figure 9).
N2 fixation was augmented when the bacterial
isolate was inoculated on Codium not treated with antibiotics.
The
antibiotic treatment apparently had I ittle or no effect on Codium
itself as production measurements conducted at the end of the experiment
indicated that plants from alI treatments were actively photosynthesizing.
20
5.0
''..c
-
I
4.0
?:
>.
'"0
01
0
3.0
w
X
1.1...
z
(\1
01
2.0
::t
1.0
2
4
8
10
DAYS
Figure 9.
Time course of the response of N fixation on Codium fragile •
2
(A) not treated with antibiotics but inoculated with N
2
fixing bacterial isolate; (8) not treated with antibiotics
and not inoculated with bacterial isolate; (C) treated with
antibiotics and inoculated with bacterial isolate; (D) treated
with antibiotics and not inoculated with bacterial isolate.
Ranges are indicated by vertical I ine through mean datum point
(three rep I icates).
21
DISCUSSION
These data suggest a symbiotic relationship between the green
macroalga Codium tragi le and an attached Azotobacter.
The I ight vs
photosynthesis and N2 fixation experiments indicate that the bacteria
may receive their reducing abi I ity from the alga.
In fact, Brinkhaus
_l
and Churchi I I (1972) noted that 0.7 to 1.3 mg glucose g dry wt
_l
hr
is excreted by Codium in the I ight, and it is known that Azotobacter
can metabolize glucose (Johnstone, 1974).
Brinkhaus and Churchi I I
(1972) calculated that as much as 40% of the carbon assimilated by
Codium may be excreted.
The released glucose could provide the reducing
abi I ity for the bacterium, and indeed, I did note an increase in
N2 fixation with glucose
addition~
vitro.
Brinkhaus and Churchi I I
(1972) also noted no glucose excretion by Codium in the dark and
similarly, I found no N2 fixation in the dark.
Azotobacter is described
as a polyphage by Mishustin and Shik 1 nokova (1971) and can use a
variety of carbon sources such as monosaccharides, disaccharides,
some polysaccharides and organic acids of the fatty and aromatic
series; possibly these compounds are also released by Codium and
uti I ized by the bacteria.
Measurements of C2 H2 :N 2 reduction ratios vary from 2.5 to 6.0 in
cell-free extracts of bacteria (Stewart et
1969; Jeng et
et
~.,
~.,
~.,
1968; Fisher and Bri II,
1969; Bergersen, 1970; Mague and Burris, 1972; Hardy
1973), but there is I ittle information avai !able from intact
I iving systems.
Considerable variations in the ratio could be
encountered due to differences between the two reactions.
Although
22
acetylene is iso-electronic with nitrogen and has similar molecular
dimensions, it is about 65 times more soluble in water than nitrogen
(Bergersen, 1970).
Experiments with Azotobacter preparations indicate
that C2 H2 and N2 evoke identical responses from the nitrogenase
system (Hardy et
~.,
1968); however, the products of acetylene
reduction do not contribute to the metabolism of whole eel I systems,
while fixed nitrogen can be uti I ized for protein synthesis (Bergersen,
1970).
Other I ines of evidence which indicate that C2 H2 and N2
behave differently as enzyme substrates are reviewed by Hardy et
(1973).
31.
Clearly, a calibration for the relationship between nitrogen
fixation and acetylene reduction should be established when comparing
nitrogen fixing systems.
This was not done in my investigation due
to the unavailability of the
15
N2 isotope, but the error may be
minimal because I examined only one nitrogen fixing system.
Dalton and Postgate (1968) suggest that the nitrogenase system
of Azotobacter is protected from damage by oxygen through augmented
respiration which would remove oxygen from the region of the nitrogen
fixing site.
When the carbon-energy source is the I imiting substrate,
Azotobacter becomes sensitive to oxygen which results in an inactivation,
but not destruction, of the nitrogenase system.
The cessation of N2
fixation in the dark by Azotobacter on Codium may be the result of
inactivation of the nitrogenase system due to depletion of carbon
energy sources and a buildup of high p0 2 values around the N2
fixing site.
In my study the average rate of photosynthesis at 100% sun I ight
in summer was about 1.7 mg C g dry wt
_l
hr
_l
Assuming, from the data
23
of Brinkhaus and Churchil I (1972), that 0.7 to 1.3 mg glucose is
released g dry wt
_1
hr
_l
, then about 16 to 31% of the carbon assimilated
at 100% I ight is released.
Wassman and Ramus (1973) report a maximum
_l
net production of 3.9 mg C g dry wt
Sound.
_l
hr
for Codium from Long Island
This is almost twice as high as my maximum value for primary
_l
production (2.0 mg C g dry wt
_l
hr
) along Cape Cod, and may be
due to the use of young plants (ca 3-5 em in length) in production
estimates by Wassman and Ramus (1973) as opposed to the use of
older plants Cca 20-25 em in length) in my production estimates.
~·
tragi le grows apically (Wassman and Ramus, 1973); therefore,
young algal plants may exhibit a higher photosynthetic capacity
per unit weight than older plants.
Low N2 fixation rates were associated with young plants of
Codium and the tip portion (distal 4-6 em) of adult Codium.
This
may be due to antibacterial activity of young algal plants and the
branched tips of adults (Conover and Sieburth, 1964), or to the fact
that there was inadequate time for dense populations of N2 fixing
bacteria. to become established on these new portions of Codium.
From the I ight vs N2 fixation and photosynthesis studies (Figure 1)
it would appear that only a smal I fraction of the carbon assimilated
by Codium is required for bacterial N2 fixation.
There was an average
3
of 3.45 x 10
pg C (net) assimilated per pg N2 fixed at 100% sun I ight.
According to Wi I son Cp. 193, 1940) 1 pg N2 is fixed per 100 pg glucose
Cor 40 pg C) consumed by Azotobacter.
Similarly, Pshenin (1963)
determined that 0.65 pg N2 are fixed per 100 pg glucose consumed by
24
Azotobacter isolated from the Black Seu.
The percentage of carbon
assimilated by Codium that is required to fix 1
with decreasing I ight intensities.
the carbon required to fix 1
~g
At
60~100,
~g
of N2 increases
30, 15, and 3% I ight
N2 is about 1.5, 2.3, 2.9 and 3.2%
respectively, of that assimilated in photosynthesis.
Thus if Brinkhaus
and Churchi I I 1 s (1972) data for glucose excretion is correct, then
a large percentage of the glucose released is not uti I ized by the
N2 fixing bacteria.
From my studies it would appear that only a very
smal I percentage of the net carbon assimilated by Codium in photosynthesis is required by the bacteria to fix atmospheric N2 •
The bacteria on Codium could provide the alga with combined N
compounds.
The maximum rates 1 of N2 fixation associated with Codium
_l
were 4.2 and 7.3
respectively.
~g
N2 fixed g dry wt
_l
hr
~situ
and~
vitro,
With a eel lular N-content for Codium of 10 ± 2.2
_l
~g
N mg dry wt
and N2 fixation occurring 12 hr per day, it would
take about 4 to 7 months to double the organic N content of Codium.
However, because Codium is a perennial plant with a I ife span of
30 months (Moe I ler, 1969), it is possible that N compounds released
by the bacteria could be of considerable benefit.
Algal photosynthesis
in North Atlantic coastal waters is I imited by the avai labi I ity of
nitrogen compounds (Ryther and Dunstan, 1971), so that any N provided
by N2 fixation could stimulate the growth of an alga.
Azotobacter
has been shown to I iberate fixed nitrogen in the form of ammonia and
amides (Newton et
~.,
1953) and some species of green macroalgae
can uti I ize organic nitrogen compounds (Lewin, 1955; Berglund, 1969).
If the bacteria on Codium release N compounds then the alga could
presumably uti I ize the released N.
25
From C/N ratios of Codium, N2 fixation rates, and Codium production
rates I have estimated the importance of N2 fixation to the nitrogen
budget of Codium.
C/N ratios, expressed on a weight basis, ranged
from 9 to 28 with a mean of 16 (n=24).
Assuming a 12 hr day and
an average production estimate for Codium of 20 mg C g dry wt
_l
this alga must obtain 1.25 mg N g dry wt
value of 0.05 mg N2 fixed g dry wt
_l
day
day
_l
_l
day
_l
_l
My
maximum~
situ
would thus provide only 4
per cent of Codium's nitrogen requirement per day.
The percentage
ranges between 2 and 7% for the lowest and highest C/N ratio found
for Codium.
This value probably underestimates the importance of N2
fixation, because N2 fixation represents a source of new nitrogen
(Dugdale and Goering, 1967) and may be repeatedly recycled by processes
of excretion, cell lysis, and grazing (Jones and Stewart, 1969; Mague
et
~.,
1974).
other compounds.
The bacteria could also aid Codium by the release of
For example, Mishustin and Shi I 'nikova (1971)
concluded that for some plants Azotobacter provides a growth stimulant
not related to N2 fixation and subsequent release of combined N
compounds.
Treatment with penici I I in G and streptomycin sulfate has the
effect of inhibiting or ki I I ing both bacteria and blue-green algae
(Vance, 1966).
The results of the antibiotic experiment indicate
that Codium can support the N2 fixing bacterial isolate.
The high
range of N2 fixation values for Codium inoculated with the N2
fixing bacteria (figure 9) is probably due to some areas of the alga's
surface being inoculated more heavily than others.
In transferring
the N2 fixing bacteria from isolation to the surface of Codium
26
no attempt was made to estimate the number of bacteria transferred
although each alga was inoculated with roughly the same area of
bacteria from the agar surfact.
Often the bacteria-plant relationships are very specific.
For
example, Azotobacter has a specific association with the rhizosphere
of the subtropical grass Paspalum notatum in South America.
Other
Paspalum species from the same locality wi I I not harbor the Azotobacter
even when it is i nocu I ated ( Dobere i ner et
~·,
1972).
In my study
I did not observe N2 fixation associated with any other macroalgae
in the Woods Hole area, and perhaps the association of this strain
of Azotobacter with Codium is specific.
From the culturing experiments and the survey conducted along
Cape Cod and Buzzard's Bay, it was apparent that N2 fixation is
depressed in the presence of available combined nitrogen compounds
and, in the field, ceases at concentrations above about 10 pg
atoms per I iter (Figure 5).
Wilson et al. (1943) demonstrated that
laboratory cultures of Azotobacter vinelandii would readily uti I ize
NHt and urea to the exclusion of molecular nitrogen.
This would be
expected since N2 fixation is energetically the most costly way
of acquiring nitrogen (Pratt, 1962).
These studies suggest that N2
fixation becomes important only when other sources of combined
nitrogen have been depleted.
There is a large range of N2 fixation values at low combined
nitrogen concentrations (Figure 5).
number of factors.
unevenly on the
This scatter may be due to a
First, the N2 fixing bacteria may be distributed
surfac~
of Codium.
Second, if the N2 fixing bacteria
27
originate from terrestrial sources its presence on Codium would,
in part, be regulated by the proximity of Codium to the source
of the bacteria.
Third, my laboratory experiments indicate that
nitrogen deficient conditions would have to persist for about
3-6 days before N2 fixation is 'turned on', so the duration of
nitrogen deficiency becomes important in
regulat~ng
the magnitude
of N2 fixation on Codium.
In the summer the average input of nitrogen at Nobska Beach
_2
on Vineyard Sound was 2.0 mg N m
_l
day
in the shallow stratum
(1-3m depth interval) and about 0.47 mg N mwaters
(3~5
m depth interval).
2
day
_l
in the deeper
In comparison, the concentration
of No;, N03, and NHt in Vineyard Sound usually become undetectable
in August.
Concentrations of particulate nitrogen in the water
_3
column at this site in summer are about 50 (range± 10) ng m
(Carpenter, unpublished data).
The nitrogen introduced by N2
fixation associated with Codium thus can be of major significance
to shallow bays such as these.
28
LITERATURE CITED
Ben-Avraham, Z. 1971. Accumulation of stones on beaches by Codium
frag i Ie. Limnol. Oceanogr. 1.§_: 553-554.
Bergersen, F. J. 1970. The quantitative relationship between nitrogen
fixation and the acetylene-reduction assay. Aust. J. bioi. Sci. 23:
1015-1025.
Berglund, H. 1969. Stimulation of growth of two marine green algae by
organic substances excreted by Enteromorpha I inza in unialgal and
axenic cultures. Physiol. Plant. 22: 1069--1073.
Bouck, G. B~, and E. Morgan. 1957. The occurrence of Codium in Long
Island waters. Bul I. Torrey Bot. Club 84: 384-387.
Brinkhaus, B. H., and A. C. Churchi I I. 1972. Primary productivity of
Codium fragile. J. Phycol. (suppl.) Q_: 15.
Conover, J. T., and J. M. Sieburth. 1964. Effect of Sargassum distribution on its epibiota and antibacterial activity. Botan. Marina
6: 147-157.
Dalton, H., and J. R. Postgate. 1968. Effect of oxygen on growth of
Azotobacter chroococuum in batch and continuous culture. J. gen.
Microbial. 54: 463-473.
Dobereiner, J., J. M. Day, and P. J. Dart. 1972. Nitrogenase activity
and oxygen sensitivity of the Paspalum notatum- Azotobacter paspal i
association. J. gen. Microbial. ]l: 103-116.
Dugdale, R. C., and J. J. Goering. 1967. Uptake of new and regenerated
forms of nitrogen in primary productivity. Limnol. Oceanogr. ~:
196-206.
Fisher, R. J. and N.J. Brill. 1969. Mutants of Azotobacter vinelandii
unable to fix nitrogen. Bact. Proc. p. 148.
Gui liard, R. R. L., and J. H. Ryther. 1962. Studies on marine planktonic
diatoms I. Cyclotel Ia nana Hustedt and Detonula confervacea (Cleve)
Gran. Can. J. Microbial. 8: 229-239.
Hardy, R. W. F., R. D. Holsten, E. K. Jackson, and R. C. Burns. 1968.
The acetylene-ethylene assay for N2 fixation: Laboratory and field
evaluation. Plant. Physiol. 43: 1185-1207.
----~----~-'
R. C. Burns, and R. D. Holsten. 1973. Application of
the acetylene-ethylene assay for measurement of nitrogen fixation.
Soi I Bioi. Biochem. 5: 47-81.
29
lssatchenko, B. 1926. Sur Ia nitrification dens lens mers.
Rend. Acad. Sci. Paris, 182: 185.
Compt.
Jeng, D. Y., T. Devanthan, and L. E. Mortenson. 1969. Components of
eel !-free extracts of Clostridium pasteurianum as required for
acetylene reduction and N2 fixation. Biochem. biophys. Res. Commun.
35: 625-633.
Johnstone, D. B. 1974. Azotobacteraceae In: R. E. Buchanan and N. E.
Gibbons (ed.), Bergey's Manual of Determinative Bacteriology. 8th ed.
Wi II iams and Wi I kins (in press).
Jones, K., and W. D. P. Stewart. 1969. Nitrogen turnover in marine
and brackish habitats. Ill. The production of extracellular
nitrogen by Calothrix scopulorum. J. mar. bioi. Ass. U.K. 49:
475-488.
Lewin, R. 1955.
33: 5-10.
Culture of Prasiola stipitata Suhr.
Mague, T. H., and R. H. Burris. 1972.
nitrogen by field-grown soybeans.
Can. J. Bot.
Reduction of acetylene and
New Phyto I • 1..!_: 275-286.
, N. M. Weare, and 0. Holm-Hansen. 1974.
in the North Pacific Ocean. Mar. Bioi. 24: 32-36.
----:---:-:----:-:--·
Nitrogen fixation
Malinowski, K. C., and J. Ramus. 1973. Growth of the greel alga Codium
fragile in a Connecticut estuary. J • Ph yeo I • 9: 102-11 0.
Mishustin, E. N., and V. K. Shi I 'nikova. 1971. Biological fixation
of atmospheric nitrogen. Penn. St. Univ. Press. University Park,
420 pp.
Moeller, H. 1969. Ecology and I ife history of Codium fragile subsp.
tomentosoides. Ph.D. thesis, Rutgers Univ.
Neish, A. C., and C. H. Fox. 1971. Greenhouse experiments on the
vegetative propagation of Chondrus crispus (irish moss).
ARL Tech. Rep. Jl.: 35 pp.
Newel I, B. S., B. Morgan, and J. Cundy. 1967.
in seawater. J. Mar. Res. 25: 201-202.
The determination of urea
Newton, J. W., P. W. Wilson, and R. H. Burris. 1953. Direct demonstration
of ammonia as an intermediate in nitrogen fixation by Azotobacter.
J. Bioi. Chern. 204: 445-451.
Pratt, J. M.
1962.
The fixation of nitrogen.
J. Theor. Bioi.
I:
251-258.
Pshenin, L. W. 1963. Distribution and ecology of Azotobacter in the
Black Sea. p. 383-391. ~: C. H. Oppehneimer (ed.), Marine
Microbiology. Springfield, Ill. C. C. Thomas.
30
Ramus, J.
1971.
Codium:
The invader.
Discovery~:
58-68.
Reinke, J. 1903. Die zur Ernahrung aer Meeres-Organismen disponiblen.
Que I lenan Stickstoff. Derdtsch. bot. Ges. £1: 371.
Ryther, J. H., and W. M. Dunstan. 1971. Nitrogen, phosphorus, and
eutrophication in the coastal marine environment. Science 121:
1008-1013.
Schol lorn, R., and R. H. Burris. 1967. Acetylene as a competitive
inhibitor of nitrogen fixation. Proc. Nat. Acad. Sci. 58: 213216.
Stewart, W. D. P. 1971. Nitrogen fixation in the sea. p. 537-564.
In: J. D. G. Costlow (ed.), Ferti I ity of the sea, Gordon and
Breach.
------------~'
G. P. Fitzgerald, and R. H. Burris.
reduction by nitrogen-fixing blue-green algae.
62: 336-348.
1968. Acetylene
Arch. Mikrobiol.
Strickland, J. D. H.; and T. R. Parsons. 1968. A practical handbook
of seawater analysis. Bul I. 167. Fisheries Res. Bd. Canada,
Ottawa.
Vance, B. D. 1966. Sensitivity of Microcystis aeruginosa and other
blue-green algae and associated bacteria to selected antibiotics.
J • Phyco I • 2: 125-128.
Wassman, E. R., and J. Ramus. 1973. Primary-production measurements
for the green seaweed Codium tragi le in Long Island Sound.
Mar. Bioi. 21: 289-297.
WI I son , P. W.
Madison:
1940. The biochemistry of symbiotic nitrogen fixation.
The University of Wisconsin Press, p. 193.
----~----~-'
J. F. Hul I, and R. H. Burns. 1943. Competition between
free and combined nitrogen in nutrition of Azotobacter. Proc. Nat.
Acad. Sci. 29: 289-294.
Yamada, N. 1961. Studies on the manure for seaweed. I. On the change
of nitrogenous component of Gel idium amansi i cultured with different
nitrogen sources. (In Japanese) Jap. Soc. Sci. Fish. 27: 953-957.