Download nitrogen fixation in an estuarine environment

LIMNOLOGY
September 1971
VOLUME
AND
NUMBER
OCEANOGRAPHY
NITROGEN FIXATION
THE WACCASASSA
IN AN ESTUARINE
ON THE FLORIDA
XVI
5
ENVIRONMENT:
GULF COAST1
Ralph H. Brooks, Jr.,” Patrick L. Brexonik,
Hugh D. Putnam, and Michael A. Keirn
Department
of Environmental
Engineering,
University
of Florida,
Gainesville
32601
ABSTRACT
Nitrogen fixation has been detected by the acetylene reduction method in the sediments
of the Waccasassa estuary, a shallow embayment on the Florida Gulf Coast. Fixation rates
in the range 1.6-15.0 ng C2H4/g sediment-hr
were found within the top 2-5cm
stratum
of sediments. Expressed in terms of equivalent
nitrogen fixed, the range was 0.64-6.0 ng
N/g-hr.
Much lower rates (0.03-0.40
ng GHa/g-hr)
were found at greater depths in the
sediment, and no fixation was observed in the flocculent
unconsolidated
l-2 cm at the
sediment surface.
All evidence indicates that the reduction of acetylene to ethylene is a biological phenomenon, directly related to the activity of nitrogen-fixing
organisms in the sediments. Nitrogen-free media produced
growths of Gram-positive
spore-forming
rods from sediments
A pure culture similar to Clostridium
sp. was isolated on nitrounder an Nz atmosphere.
gen-free media from Waccasassa sediments and was shown capable of nitrogen fixation
by the acetylene reduction method.
INTRODUCTION
The process of nitrogen fixation has significance for world geochemistry as well
as for localized areas within the biosphere.
Hutchinson (1944) noted that without this
continuous supply fixed nitrogen levels
would be dissipated within a period of
several million years. Certainly nitrogen
fixation may allow the continuation
of
organic production
when fixed nitrogen
supplies are depleted, as has been well
established for aquatic environments.
1 Parts of this paper are from a Ph.D. thesis
(University
of Florida)
by R.H.B.
This research
was supported by Federal Water Quality Administration
research grants 16010DCK
and 18050
DTK and training grant 5TOl-UI-01029-08.
‘Present
address:
Pacific Gas & Electric
Co.,
245 Market St., San Francisco, Calif.
94106.
LIMNOLOGY
AND
OCEANOGRAPHY
Apparently
blue-green
algae are the
dominant
agents of such processes in
aquatic environments (e.g., Dugdale et al.
1959, 1964; Dugdale and Dugdale 1962;
Goering and Neess 1964; Goering et al.
1966). However, Brezonik and Harper
(1969) have reported bacterial nitrogen
fixation in the aphotic and anoxic zones of
several lakes. Further studies by Keirn
and Brezonik (1971) show NP fixation in
lacustrine sediments from a variety of
Florida
and Guatemala lakes. Stewart
(1968) reported nitrogen fixation at the
thermocline of a Norwegian lake correlated with the presence of photosynthetic
bacteria. In situ bacterial fixation of nitrogen has not previously been reported
within oceanic or estuarine waters or sediments although both Axotobacter and NZ701
SEPTEMBER
1971, V. 16(5)
702
R. I-1. BROOKS,
JR.,
I?. L.
RREZONIK,
fixing CZostridium have been isolated from
the Black Sea (Pshenin 1959, 1963). A
wide variety of anaerobic and facultative
bacteria arc known to fix nitrogen in pure
cultures. Many of these species are likely
to be fo,und in organic sediments, for example Desulfovibrio
desulfuricans
( Sisler
and ZoBell 1951), methanogenic bacteria
( Pine and Barker 1954)) and various clostridia. In addition, methane-oxidizing
bacteria (Pseudornonus)
are known to fix
nitrogen (Davis et al. 1964; Coty 1967) and
may inhabit the sediment-water
interface.
One might reason that organic estuarine
sediment is an unlikely habitat for nitrogen-fixing
organisms since this environment is normally considered to be rich in
the fixed nitrogen
Howcvcr,
nitrogen.
measured by chemical analysis is not necessarily identical with the fraction that is
biologically
available. For example, sedimcnt ammonia may be sorbed onto clays
and be essentially unavailable to microorganisms. Stewart ( 1969) has recently
shown that algae will continue to fix nitrogen in the presence of modcrate concentrations
of ammonia, although
high
ammonia lcvcls eventually repress synthcsis of the nitrogenase system. The fact
that many bacteria are capable of nitrogen fixation is not proof that they actually
fix nitrogen in any given environment but
it dots suggest that this capability is useful
for the growth and survival of these organisms in som,e environments.
Stewart et al. (1967) first indicated the
feasibility of using acetylene reduction as
an index of nitrogen fixation in the field.
The rate of ethylene production when molecular nitrogen is removed from a sample
and purified acetylene is added is a measure of the rate of nitrogen fixation. The
chief advantages of the method over the
conventional 15N technique are simplicity
an d sensitivity, with incubation times as
short as 30 min, making the method cspecially adaptable for use in field surveys
where rates may be low.
The Waccasassa estuary, an unpolluted
embayment on the Gulf Coast elf Florida
(Fig. l), is a shallo,w, well-mixed bay with
I-I. D. PUTNAM,
AND
M.
A. KEIRN
small planktonic and benthic populations.
Putnam (1966) has shown that low nitrogen and phosphorus lcvcls limit primary
production in the area.
MATERIALS
AND
METHODS
Nitrogen fixation was determined by an
adaptation
of the acetylene reduction
method (Stewart et al. 1967) previously
described by Brezonik and Harper (1969).
Incubations were conducted in glass serum
bottles (approximate capacity, 70 ml) fitted with rubber serum caps. Exact bottle
volume was dctcrmincd by mass of water
content. Purified grades of gases and high
purity gas mixtures were obtained commercially (Mathcson Co.). All water samples were incubated under an artificial
aerobic atmosphere coinsisting of 20% 02,
0.04% COZ, balance Ar. Raw water samples were incubated in situ; some water
samples were concentrated 2O-fold by continuous centrifugation
and were incubated
in the laboratory
(at about 2OC. under
Sylvania daylight type fluorescent lighting). Sediment was incubated in the laboratory (in the dark at ca. 2OC) by adding
3040 ml of a sediment slurry to the serum
bottle and purging either with the artificial aerobic atmosphere or with pure hclium to maintain anoxic conditions.
The incubation
bottles were purged
with gas through a manifold of polyethylene tubing connected to a 22-gauge hypodermic needle that exte.nded to the bottom
of the bottle, Gases were vented by a
second needle positioned above the watersediment phase. After flushing with the
appropriate gas phase, we too,k care to
maintain
atmospheric
pressure through
evacuations corresponding to the volume
of gas (normally 5 cc of acetylene) added
to the incubations.
Incubations lasted for
0.54 hr. Reactions were terminated with
trichloroacetic
acid ( l-2 ml of 50% aqucous solution) or mercuric chloride (2-4 ml
of saturated solution).
To avoid possible
leakage, serum caps were scaled with silicone grcasc.
Ethylene was separated from acetylene
NITROGEN
FIXATION
IN
WACCASASSA
703
ESTUAnY
WACCASASSA
GULF
OF
SCALE IN
KILOMETERS
MEXICO
‘45’
FIG.
1.
The Waccasassa
estuary
embayment
and the ethylene produced was measured
by gas chromatography.
.A Varian-Acrograph model 600D gas chromatograph
with hydrogen flame ionization
detector
was used with a 9 ft x l/s inch (2.7 m x
0.3 cm) Porapak T column, carrier gas
(high purity nitrogen)
flolw rate of 25
cc/min, and column temperature of 50C.
Peaks were located by using purified
gasles, and ethylene peak areas were calibrated by means of purified
ethylene.
Aqueous fixation is expressed as ng N
and its location
on the Gulf
Coast of Florida.
fixed/liter-hr;
for sediments the rates arc
in terms of ng N fixed/g sediment (dry
wt basis) -hr.
Controls were run with each cxperiment by adding TCA to samples before
acctylcne addition.
They were necessary
because of rather wide variations of background ethylene in the acctylcnc. Ethylenc levels were especially high in new
tanks and declined with use. To avoid
complications
arising from high backgrounds we found it necessary to waste the
704
1~. H. BROOKS,
TABLE
JR.,
P. L. BREZONIK,
1. Acetylene
reduction
rates in Waccasassa estuary water, 13 July 1968”
Low tide
Station?
I
II
IIigh
Surface
Middle
13ottom
39
34
77
58
51
136
Surf ace
Middle
Bottom
53
41
-
84
107
184
tide
+ Results in ng ethylene/liter-lx;
values are means of
duplicate incubations.
-1 Both stations located in the embayment in 1.5 m of
water at low tide and about 2.1 m at high tide.
first third of a tank. Some workers have
reported abiological production of ethylenc in high organic soils when TCA is
added, but we found no evidence for this
in Waccasassa sediments. Control ethylene peaks were always low to undetectable
when good acetylene was used. Areas of
the control peaks were subtracted from
the peak areas of the incubated samples
to determine the rate of biological ethylenc production.
RESULTS
Low rates of ethylene production were
found within the water column and sediments of the Waccasassa Estuary using in
situ and laboratory incubations. Although
acetylene reduction was consistently found
within the bottom sediments, it was detected in embayment water samples only
once.
In a series of in situ incubations during
July 1968, ethylene production was noted
within the water column with highest activity near the botto,m (Table 1). Subscqucntly, in November 1968 and in May
1969, in situ incubations gave no evidence
for acetylcnc reduction. In July and November 1968 and February 1969, solids in
the estuary water were concentrated about
20-fold by continuous centrifugation
to
achieve a total Kjeldahl nitro*gen of about
0.23-0.37 mg N/incubation
bottle; the concentrated water samples were exposed to
acetylene during several laboratory incubations and no ethylene production could
be detected. All of the water column stud-
I-1. D. PUTNAM,
AND
M.
A.
KElRN
its included high and low tide water and
used 4-6-hr incubations,
It is doubtful
that the single occurrence of fixation in
the water reflected a difference in the
type od water present at that time. Salinity
in the embayment varies between 17 and
24%0 at high tide and 12 and 21%0 at low
tide. Except under unusual circumstances
(e.g., hurricanes) seasonal salinity effects
within the embayment are rather small.
Effects arc of course more pronounced in
the upper (pre-embayment)
section of the
estuary, where summer rains cause considerablc freshening of the river water;
but this study was confined to the embayment, which by virtue of its size is much
less affected by summer runolff.
The range of 51-184 ng ethyIene/literhr found in July 1968 can be expressed in
terms of equivalent nitrogen fixed if we
assume the theoretical ratio of 1.5 moles
of ethylene produced per mole of ammonia
fixed. The reduction of acetylene to ethylene requires two electrons and the rcduction of Na to 2 NH3 requires six, so the
rate of ethylene production should be 1.5
times the rate of ammonia production,
assuming that electron transfer is the ratelimiting step. Using the acetylene reduction and 15N techniques, Stewart et al.
(1968) measured ratios ranging from 1.41.8 for Aphanixomenon;
Schollhorn and
Burris (1967) repo,rted a value of 1.25 folr
Axotobacter and Clostridium.
Conversion
of the data in Table 1 to equivalent nitrogen fixed (assuming a molar ratio of! 1.5)
indicates a range of 16.9-61.3 ng N fixed/
liter-hr.
Although analysis of seasonal aspects of
sediment fixation was not our purpose, nitrogen fixation was found in the estuarine
sediments on every sampling
occasion
throughout the year. An initial experiment
with the sediment to determine the optimal length of incubmation showed that the
rate of acetylene reduction was appro,ximately linear up to, 1 hr; with longer
incubations, rates were lower and more
variable ( Fig. 2). The mean for six sediment replicates incubated to assess the
precision of the techniques was 11.5 ng
NITROGEN
FIXATlON
IN
WACCASASSA
incubation
FIG.
2.
Effect
of incubation
Time
time on the amount
ethylene/g
sediment&r,
with a relative
standard deviation of 3.6% (Table 2).
Rates of cthylcnc production in cmbaymmt sediments for all olur determinations
(over 60) ranged from an undetectable
amount to 15.0 ng ethylene/g dry wt scdiment-hr.
A distinct layering was found
within a typical sediment core ( Fig. 3).
In the flocculent, unconsolidated
l-2 cm
at the core surface, no acetylene reduction
was found at any time. Significant rcduction rates were consistently noted in the
next 2-5 cm of consollidated gray-black
ooze, ranging from 1.615.0 ng CzH4/g
sediment-hr.
From 5-20 cm the typical
2.
Results of replicate
incubations
Ethylene
ng
sediment
55.9
51.2
60.5
58.2
51.2
53.4
produced
by estuary
sediment.
core consists of coarse organic material
overlying the limcrock substratum; acetylcac reduction was always found in this
zone, but the rates ( 0.03-0.40 ng C&14/g
sediment-hr)
were low compared with
those in the upper portions of the core.
An arcal survey elf the embayment
indicated that acctylcne reductioa is a
consistent phcnomcnon in the upper scdimcnts. Duplicate cores were taken at 8
Ethylene
Production:
nannograms
2.5””
(g
dry wt 1.’ hr -’
None detectable
1.62
- 15.0
produced
rig/g sediment
t
5-20 cm
4.827
4.631
4.971
4.961
4.582
4.760
(Hours)
of ethylene
0.2
TABLE
705
ESTUARY
11.6
11.1
12.2
11.7
11.2
11.3
Mean = 11.5
s* = 0.17 s = 0.41
0.032.0.40
L
FIG. 3.
Section of sediment core illustrating
variation
of acetylene
reduction
with
depth.
Range of ethylene production
represents a minimum of six determinations
in each layer.
706
R. H. RROOKS,
JR.,
P. L.
UREZONIK,
Distribution
of acetylene
reduction
FIG. 4.
within sediments of the Waccasassa embayment.
Data from 2-5cm
segments of duplicate
cores
at 8 stations. Station II was in a mudflat.
stations, the upper 2-s-cm portion of each
was blended to obtain a homolgcneous
sample, and acetylene reduction rates were
determined ( Fig. 4). The range was from
4.9-15.0 ng ethylene/g
sediment-hr, but
most of the values were near the mean rate
of 9.2. Expressed in terms of the equivalent amount of nitrogen fixed (assuming
the theoretical ratio of 1.5 moles C&I* produccd per mole NH3 fixed), the results
indicate a mean and range of 3.07 and 1.65.0 ng N/g dry wt-hr.
Several experiments were undertaken to
evaluate the environmental
conditions for
fixation in sediments and to define the
agents of fixation. The effect of a nitrogen-fret oxygen atmosphere vs. an anoxic
( helium) environment was studied in one
experiment; in all but oae of five samples,
the rate of ethylene production was higher
II.
D. PUTNAM,
AND
M.
A.
KEIRN
under a helium atmosphere, but the differences were not so great as would be
expected if strict anaerobes were the fixing
agents. Probably the organic sediments cxerted a sufficient o,xygen demand to maintain anoxic microzones in the samples oven
in an oxygen atmosphere.
Exposure of incubating samples to light
decreased the acetylene reduction activity;
the me,an rate of cthylcne production in
five replicates incubated in the light was
only a third of the mean value for those
incubated in the dark. The reasons for this
cannot be fully explained; howcvcr, it is
well known that the activities of solme
microorganisms can be inhibited by visible
radiation.
Since the agents of ethylene production
(hence nitrogen fixation) in the sediments
arc presumably anaerobic or facultative
bacteria, the addition of easily assimilable
carbonaceous substrate to sediment samples should enhance their activity. Scvcral
experiments have given somewhat conflicting results in this regard. In an early
experiment acctatc gave apparent stimulation of acetylene reduction at concentrations of 0.02 and 0.2 M but glucose gave
no response. However no acetate stimulation was found in three later experiments
in the range 10m4to 10-l M; in fact 10-l
M acetate and butyrate
actually inhibited
fixation by about 30%. No evidence for
glucose stimulation o’r inhibition was found
in any of the four enrichment experiments
in the concentration range 10m4to 10-l M,
but sucrose gave definite stimulation (SO100% incrcascs over controls) in each of
the three experiments in which it was
added. Similar responses to added organic
substrate have been found for freshwater
lake sediments by K&n
and Brezonik
(1971).
Experiments evaluating the impact of inhibitors on acetylene reduction showed it
was completely inhibited in 30-40 ml of
scdimcnt slurries by 1 ml of 50% trichloroacetic acid or 3 ml of saturated mercuric
chloride solution. Acetylene is a competitive inhibitor of Na (Schollhorn and Burris
1967), so the convcrsc should be true, i.e.,
NITROGEN
FIXATION
IN
WACCASASSA
707
ESTUARY
11 I31 I [Substrate] -’
Classical
Lineweaver-Burk
plot for
[S] is substrate (reactant)
competitive
inhibition.
concentration;
v is the rate of the enzyme catalyzed reaction, and I represents an enzyme inhibitor with concentration
I2 greater than 11.
FIG.
5.
added molecular nitrogen should reduce
the rate of acetylene reduction according
to the competitive enzyme inhibition
pattern (see Mahler and Cordes 1966). Thus
a reciprocal (Linoweavcr-Burk)
plo,t of reaction velocity (ethylene production rate)
vs. substrate
(acetylene)
concentration
should be linear, and added inhibitor (m.olecular nitrogen) should give the classical
competitive inhibition
plot ( Fig, 5). To
verify this for Waccasassa sediments, two
sets of samples were set up, One series
was exposed to various concentrations of
acetylene in a helium (nitrogen-free) atmosphere; in another series, samples elf the
same sediment were exposed to various
FIG.
6.
Ethylene
production
vs. acetylene
concentration
for Waccasassa sediment.
Samples
were incubated for 1 hr; 30 ml of sediment slurry
was in each 70-ml serum bottle.
Helium atmosphere = 0; N2 atmosphere = q .
It
[S]
kdbottle)”
plot of data from
7. Lineweaver-Burk
Fig. 6 showing competitive
inhibition
of ethylene
production
by Na in Waccasassa sediment.
Helium atmosphere = 0; N2 atmosphere = 0.
FIG.
acetylene concentrations in a 70% Ng-30%
CO2 atmosphere. A plot of ethylene production rate vs. acetylene concentration
for the two series ( Fig. 6) indicates typical cnzymc or saturation kinetics given by
the Michaelis-Mcnten
model,
A Lineweaver-Burk
plot of the data (Fig,. 7)
shows the classical competitive inhibition
pattern. The maximum velocity (V,,,)
as
obtained from the y-intercept of the Linewcavcr-Burk
plot is approximately
the
same for both curves, implying that high
concentrations of acetylene negate the inhibitory effect of Nz.
The organisms most likely to be responsible folr biological nitrogen fixation in the
anoxic etsuarine scdimcnts are anaerobic
bacteria of the genus Clostridium.
An enrichment and isolation procedure for such
organisms was used on the Waccasassa
sediments. A sample from the 2S-cm
depth was heated at 80C for 10 min to
destroy vegetative forms and then inoculated into a nitrogen-free salt medium containing 4 g/liter of one of the follolwing
carbon sources : acetate, maltose, mannitol, sorbitol, or sucrose. All except acetate
have been reported as fcrmentablc carbon
sources for Clostridium pasteurhum.
After incubation at room temperature ( 222%) under a pure nitrogen atmosphere
708
R. 1-I. BROOKS,
JR.,
P. L. BREZONIK,
for 3 days, growth was apparent (by increased turbidity and gas production)
in
all media except that containing acetate.
Most rapid growth occurred initially with
mannitol, but in subsequent transfers sorbitol gave the quickest response. A test fo’r
acetylene reduction activity in the mannito1 culture gave positive results. Gram
stains showed an abundance of Grampositive rods, many showing subterminal
swellings
characteristic
of developing
spores, accompanied by a few Gram-variable coccoid forms.
The enrichment cultures were incubated
5 more days to prolmotc sporulation, then
heated to 80C for 10 min and transferred
to basal salts media containing so’rbitol.
Transfers were incubated under an NZ atmosphere at room temperature for 48 hr,
and another .transfer was made to similar
media. Following incubatioa for 5 days,
a third transfer was made. Micro-Kjcldahl
analysis of each batch of fresh media
showed no detectable ammonia or organicN. A test for acetylene reduction on the
eight bo,ttlcs ( duplicates of the four original carbon sources showing growth) frosm
the third transfer gave positive results for
three bottles.
Streak plates were made onto the nitrogen-free basal salts medium plus sorbitol
in 2% agar from the incubated material of
the third transfer, and the plates were
incubated for 72 hr in an anoxic jar (Nz
atmosphere). Colonies on the plates were
all similar and not colored. Ten colon&
of Gram-positive rods were picked from
plates streaked with m,cdia from bo,ttlcs
that had exhibited
acetylene reduction
and transferred to, basal salts plus sorbitol. Each isolate was grown under both
aerobic and anaerobic (Nz atmosphcrc)
conditions. Four co,lonics grew only anacrobically; the remainder grew under both
conditions. The latter were tcstcd for acetylene reduction and gave negative results.
The four obligate anaerobic isolates were
also tested for acetylene reduction; three
showed positive rates.
The final isolates have the charactcristics of a Clostridium culture: Gram-posi-
II.
D. PUTNAM,
AND
M.
A.
KEIRN
tive, strictly anaerobic, large rods that
ferment sugars and can fix molecular nitrogen. Further physiolo,gical tests would
of course bc necessary to ,cstablish the
specific identity of the cultures. Since the
taxonomy of marine and estuarine anacrobcs is not highly developed, to place the
cultures in a particular
genus would bc
questionable.
Nonetheless, the work has
demonstrated that “clostridia-like”
nitrogen-fixing bacteria arc present in Waccasassa estuary scdimcnts. The data do not
exclude the possibility
that other nitrogen-fixing
forms are present since the
isolation method used was selective for
these bacteria.
DISCUSSION
Evidence for low rates of bacterial nitrogen fixation has been found in the
Waccasassa embayment by the acctylcne
reduction technique. With incubations of
1 hr or less the prccisioa of the test was
very good, with a relative standard deviation of about 4%. The decrease in activity
with incubation
times longer than 1 hr
may be rclatcd to bottle effects or to the
imposed atmospheric changes inherent in
the proccdurc.
Evidence for nitro’gcn fixation has been
confined chiefly to the sediments, with
highest rates found within the top 2-5 cm.
Expressed in terms of ammonia nitrogen
fixed, rates in this zone ranged from 1.65.0 ng N/g sediment-hr. Acetylene reduction activity has never been found within
the aerobic interfacial zone. It was found
only once within the well-oxygenated
water column, suggesting the possible occurrence of bacterial populatioas within the
microienvironment
of resuspended scdimerits; the distribution
with depth in the
water column and the Eailurc to dctcct
acetylene reduction under olthcr conditions
would support such a relationship.
We have sufficient evidence to conclude
with reasonable assurance that the acctylene reduction activity in the sediments
is directly related to nitrogen-fixing
organisms. All lines of evidence point to
NITROGEN
FIXATION
IN
ethylene production as a bio,logical phenomenon; bolth trichloroacctic
acid and
mercuric chloride complctcly inhibit acetylenc reduction, Added organic substrate
increased the rate of ethylene proiduction.
The fact that Nz acts as a competitive inhibitor
of acctylcnc reduction
strongly
suggests that ethylene is prolduced by nitrogenase. It should be noted that Burris
(1969) doubts his carlicr conclusion (Schiillhorn and Burris 1967) that acetylcnc is a
competitive inhibitor and now thinks it to
bc noncompetitive
[in which the rcciprocal lines (Figs. 5 and 7) intersect to1 the
left of the ordinate].
Differentiation
bctwecn these two casts is not always simplc, especially when the intersection point
is very near the ordinate.
The data in
Fig. 7 are not sufficient to, rule this possibility out, but this does not negate their
significance
in supporting
a bio’logical
mechanism for acetylene reduction, in the
sediments. Nitrogen-free media produced
growths of Gram-positive
spore-forming
rods frolm sediments under an atmosphere
of pure nitrogen. A pure culture similar
to Clostridium
sp. was isodated from the
scdimcnt and shown capable of nitrogen
fixation by acctylcnc reduction.
A rough estimate of the total amount of
nitrogen fixed in the 2-5-cm layer of scdimcnts in the Waccasassa can be obtained
from the data presented in Fig. 4. Assuming that the mean value of 3.07 ng N
fixed/g scdimcnt-hr is a reasonable estimate o,f the nitrogen fixatio,n rate in this
stratum of scdimcnt throughout the estuary, the amount of nitrogen fixed on an
annual basis is 37 rug N/cm2-yr or for the
entire 7-km2 estuary, 2.6 X 10” kg N/yr.
Thcsc values are based on an avcragc
sediment dry weight of 0.455 g/cm3 in
the 2-5-cm zone. These extrapo,lated valucs arc obviously rather crude, but they
serve to illustrate the point that the scemingly low rates found in the sediments in
fact rcprcscnt significant amounts of nitrogen fixed on an annual basis,
Because sediments are normally thought
to be cnrichcd in nutrients, we wcrc somcwhat surprised at our initial results indi-
WACCASASSA
ESTUARY
709
eating nitrolgen fixation
in Waccasassa
estuary sediments. Free ammonia was dctermined on sediments from 14 widely
scattered stations in the estuary (Brooks
1969); a range of 0.01-0.37 and mean of
0.06 mg NH3-N/g sediment was found.
Not all this ammonia would necessarily be
available to microorganisms; much or perhaps all of it could be loosely sorbed to
solid material in the sediment. Some evidence that this occurs was reported by
Broloks ( 1969)) who found that this sedimcnt contains 3-5% clay mineral and tends
to sorb ammonia rapidly from solution.
Algal fixation
of nitrolgen has been
known to occur in natural waters for a
long time (c.g., Hutchinson 1941; Stowart
1965; Stewart ,et al. 1967). Presence of
bacteria capable of fixing N2 in aquatic
environments
has also1 been kno’wn for
some time (e.g., Pshenin 1959, 1963). However the latter studies reported only laboratory isolations and gave no evidence
for in situ fixation.
Evidence for in situ
nitrogen fixation in lake waters by heterotrophic and photosynthlctic
bacteria has
been recently
reported
(Brezonik
and
Harper 1969; Stewart 1968). The prcscnt
report cxtcnds the rcportcd occurrence of
nitrogen fixation in nature to the scdiments underlying
natural waters. However, the phenomenon is probably not important as a nitrogen source to the overlying waters because of the low rates
found and the locatio,n of activity in compacted scdimcnts.
REFERENCES
BREZONIK, P. L., AND C. L. HARPER.
1969.
Nitrogen
fixation
in some anoxic lacustrine
cnvironmcnts.
Science 164: 1277-1279.
BROOKS, R. II., JR.
1969.
In situ nitrogen
dynamics
within
an estuarine
embayment.
Ph.D.
thesis,
Univ.
l?lorida,
Gainesville.
229 p.
BURRIS, R. H.
1969. Progress in the biochemistry of nitrogen
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