APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1981, p. 1413-1418
Vol. 41, No. 6
Nitrogen Fixation (Acetylene Reduction) Associated with
Decaying Leaves of Pond Cypress (Taxodium distichum var.
nutans) in a Natural and a Sewage-Enriched Cypress Dome
FORREST E. DIERBERGt* AND PATRICK L. BREZONIK
Center for Wetlands and Department ofEnvironmental Engineering Science, University ofFlorida,
Received 27 October 1980/Accepted 30 March 1981
Surface litter from a natural and a sewage-enriched cypress dome in north-
central Florida showed a pronounced seasonal pattern of nitrogenase (acetylene
reduction) activity associated with seasonal leaf fall from deciduous trees in the
domes. Samples ofpeat from cores indicated negligible nitrogenase activity below
the surface layer. Integrating the monthly rates ofnitrogen fixation (based on the
theoretical molar ratio of 3:2 for C2H4/NH3) yielded 0.39 and 0.12 g of N/mi2 per
year fixed in the litter of the natural and sewage-enriched domes, respectively.
The nitrogen fixed in the first 3 months after leaf fall in the natural dome
represented about 14% of the nitrogen increment in the decomposing cypress
leaves, but fixation contributed a negligible amount of nitrogen (<1%) to decom-
posing litter in the sewage-enriched dome.
Cypress domes are small subcircular swamps
that are found throughout the pine-palmetto
woodlands ofthe southern Atlantic and the Gulf
Coastal plain. The domes vary in area from
about 1 ha to more than 10 ha (usually less than
5 ha) and are a common geomorphological land-
forn in the region. Odum et al. (18) proposed
using cypress ecosystems as an alternative to
conventional tertiary treatment of sewage ef-
fluent, and feasibility studies on this proposal
have been under way near Gainesville, Fla., for
the past 6 years. Ewel and Odum (5) recently
concluded that the use of cypress wetlands for
tertiary treatment of sewage is favorable for
several reasons, including: (i) lower cost, com-
pared with physicochemical methods; (ii) better
filtration and less ecosystem change, compared
with upland disposal; and (iii) higher adaptabil-
ity to high nutrient and low dissolved oxygen
levels, compared with disposal into rivers and
lakes. Recycling treated sewage into cypress
wetlands can save waste treatment costs and
maintain a high-quality timber product. This
alternative for treatment of sewage effluent and
storn water runoffis being considered on a case-
by-case basis in Florida.
The disposal of treated sewage into cypress
swamps stimulated studies of nutrient cycling
processes in natural as well as sewage-affected
t Present address: Department of Enrivonmental Science
and Engineering, Florida Institute ofTechnology, Melbourne,
cypress wetlands. Since natural cypress domes
receive a limited supply of nutrients from exter-
nal sources, questions about internal nutrient
cycling processes are of interest.
Several investigators have reported that the
relative amount of nitrogen in leaf detrital sys-
tems increases during in situ decomposition (2,
4, 7, 13, 15, 17). The increases were frequently
preceded by decreases in nitrogen content dur-
ing the first few days of immersion, presumably
by leaching. Little work has been reported on
the role of nitrogen fixation in increasing nitro-
gen concentration. Howarth and Fisher (11) re-
ported nitrogen fixation (acetylene reduction)
associated with decomposing sugar maple leaves
in laboratory stream microecosystems, but they
found that fixation was an insignificant source
of nitrogen for the litter. Gotto and Taylor (8)
stated that nitrogen fixation in decomposing
leaves of the red mangrove (Rhizophora man-
gle) may contribute significantly to observed
nitrogen increases, but they did not provide a
quantitative estimate. This paper examines the
contribution of N2 fixation in replenishing the
nitrogen initially lost from decaying cypress
leaves in natural and sewage-enriched cypress
domes and evaluates the effects of disposal of
treated sewage on the decomposition ofcypress
MATERIALS AND METHODS
Study sites. The study sites were two cypress
domes near Gainesville, Fla. One cypressdome (1.05
DIERBERG AND BREZONIK
ha), located on a large pine plantation owned by the
Owens-Illinois Corp., received secondary sewage ef-
fluent (2.5 cm/week) from a 114-m3/day extended-
aeration package plantwhich served a 155-unit mobile
home park adjacent to the site. The inorganic nitrogen
content ofthe effluent averaged 10.1 mg ofN per liter,
and the organic N content averaged 7.2 mg/liter. A
larger dome (4.2 ha), located about 17 km northeast in
the Austin Cary Memorial Forest, served as a "natu-
ral" control whose water levels fluctuatedaccording to
the normal hydroperiod.
Cypress leaf decomposition. Freshly fallen cy-
press leaves were collected from the two domes in
early November 1977. Fallen litter harvested from
cleared surfaces on boardwalks, platforms, and utility
shed roofs in these sites was stored in paper bags at
room temperature until January 1978. Then 30 to 45
g (air-dry weight, nearest 0.1 g) was enclosed in (2 by
2 mm mesh) fiber glas screen bags (22 by 28 cm). On
1 January 1978, 40 bags containing litter (8.1 mg ofN
per g, dry weight) from the natural dome were placed
in a pool (1.5 m deep) of that dome. Forty bags
containing litter (15.0 mg ofN per g, dry weight) from
the sewage dome were placed near the center of the
sewage dome. The litter bags were not placed at the
edges of either dome, but Deghi found no significant
decomposition rate differences between cypress leaf
litter placed at the center and litter placed at the edge
(G. S. Deghi, M.S. thesis, University of Florida,
The litter bags were collected in groups offive from
each site after 15, 29, 58, 114, 205, and 297 days of
incubation. The contents were removed within 24 h of
collection; weights were determined after the contents
were dried at 67°C for at least 5 days, and the percent
weight loss was calculated for each bag.
Nitrogen analysis. Nitrogen concentration was
determined by the micro-Kjeldahl method after the
samples were ground in a Wiley mill (1). Duplicate
samples were within 5%, and coefficients of variation
for primary standards were within 5%.
Acetylene reduction assay. Nitrogenase activity
in litter samples from the domes was determined by
the acetylene reduction method (10, 20). Acetylene
was generated immediately before use from calcium
carbide and water, and samples were analyzed for
ethylene by injecting 0.5 ml of gas into a Varian-
Aerograph model 600 D gas chromatograph with a
column (2.7 m by 0.3 cm) packed with Poropak R and
a hydrogen flame ionization detector. Nitrogen was
the carrier gas (flow rate, 17 ml/min), and the column
temperature was 56 to 57°C. Because of problems
associated with the use of metabolic poisons in termi-
nating acetylene reduction activity (12, 19, 21), chro-
matographic analysis was performed immediately
after incubation. In all assays, controls without C2H2
were set up; however, these almost always proved to
be negligible. Ethylene contamination ofthe acetylene
was always known and allowed for in the final calcu-
Surface litter sampling and assay procedures.
Samples of surface litter and standing water were
obtained monthly by submerging a 22- or 70-ml serum
bottle to the litter surface, tuming it upright, and
allowing bottom water to displace the air inside. Or-
ganic matter (mostly cypress and black gum leaflitter)
was then inserted into the bottles while they were still
submerged, displacing the bottom water inside the
bottle. The bottles were brought to the surface and
immediately capped with a rubber serum bottle stop-
per so that the organic matter would not be exposed
to air. The bottles were transported back to the labo-
ratoryon iceandkeptrefrigerated at4C untilinjected
with acetylene (within 5 h of field collection). A high
percentage of aqueous phase was maintained (-80%)
to avoid exposing the acetylene-reducing organisms to
more oxygen than that present in the bottom waters
and to increase the sensitivity of the acetylene reduc-
tion method (6). Because of a cover of floating plants
(mainly duckweed, Lemna minor), surface water in
the sewage-enriched dome normally had low levels of
dissolved oxygen (0.0 to 0.8 mg/liter). The natural
dome did not have a plant cover, and dissolved oxygen
levels were higher, although still below saturation (0.2
to 6.8 mg/liter). Air and acetylene were injected (3 ml
of air followed by 10 ml of acetylene) to give a final
concentration ofdissolved acetylene of0.15 ml ofC2H2
per ml of water. The gases were injected with a 10-ml
syringe, and displaced water was vented through a
hypodermic needle inserted into the stopper of the
serum bottle. This procedure maintained a constant
pressure and did not expose the inside ofthe bottle to
more air than that which was injected. The bottles
were then shaken vigorously by hand for 15 to 30s to
equilibrate the vapor and aqueous phases and imme-
diately incubated at in situ water temperatures in the
dark for 3 to 25 h. At the end ofthe incubation period,
the serum bottles were again shaken vigorously for 1
min before 0.5 ml of gas phase was withdrawn for
measurement of ethylene content by gas chromatog-
raphy. Corrections for ethylene solubility in equili-
brated closed systems were made according to Henry's
Peat depth profile. Two polyvinylchloride cores
were obtained from the natural dome with a piston
corer in March and April 1979 to determine vertical
variations of acetylene reduction activity in the peat.
The cores (with overlying water) were sealed by rub-
ber stoppers to prevent exposure to the air, returned
to the laboratory, and extruded within 3h ofsampling.
Horizontal slices (2 cm thick) were obtained from
depths of 0 to 2, 3 to 5, 6 to 8, and 8 to 10 cm by
pushing the sediment up the core tubes with a piston
and slicing off the sediment at the top. Each sample
was divided into four subsamples, three ofwhich were
incubated with C2H2. The fourth subsample was in-
cubated without C2H2 as a control for the study of
endogenous production of C2H4. Both control and
experimental bottles were purged with He before acet-
ylene was added to maintain anoxic conditions during
Surface water was assayed regularly to determine
the amount of fixation in the water used to prepare
peat and litter samples for acetylene reduction asay.
Ethylene production associated with leaf
litter. Acetylene reduction activity remained
low (<2.2 nmol ofC2H4 per g [dry weight] per h)
APPL. ENVIRON. MICROBIOL.
NITROGEN FIXATION AND DECAYING LEAVES
in both domes from August 1978 until January
1979, when it increased in the natural dome,
reaching a high of 36.9 nmol of C2H4 per g (dry
weight) per h in March (Fig. 1). After March,
the rates decreased in the natural dome but
increased somewhat in the sewage dome until
June. Nitrogenase activities in both domes re-
turned to the initially low levels by August 1979.
At no time were the measured rates from the
sewage dome higher than the rates in the natural
dome. Integrating the monthly rates over the
year of measurement yielded 1.01 mg ofN fixed
per g (dry weight) per year in the litter of the
natural dome and 0.21 mg of N fixed per g (dry
weight) per year in the litter of the sewage-
Rates ofacetylene reduction were linear, with
no lag period over the range of incubation pe-
riods employed. The measured rates apply only
to the surface layer of submerged litter on the
swamp floor; core sections from below the sur-
face produced negligible amounts ofethylene on
both occasions when the cores were taken. As-
says ofthe standing water (collected at the peat-
water interface) consistently showed no ethyl-
Calculations were made to relate the observed
acetylene reduction to the quantity of nitrogen
fixed, using the following assumptions. Most of
the observed nitrogen fixation occurred in sur-
face litter from the most recent litter fall. Ac-
cording to Deghi (M.S. thesis), leaf litter fall
(average of a 2-year period) is about 390 g (dry
weight) per m' per year for the natural dome
and 590 g (dry weight) per m2 per year for the
sewage dome. The surface litter had an average
specific gravity of 0.098 g/cm3 ± 0.009 standard
deviation (n = 5). Thus, the extrapolated depth
of litter fall in 1 year was 0.39 cm for the natural
dome and 0.60 cm for the sewage dome. It was
assumed that samples of the surface litter taken
for acetylene reduction assay approximated
those depths. Finally, the theoretical molar ratio
of 3:2 for C2H4/NH3 was assumed (20), yielding
a factor of 9.3 ng ofN fixed (as NH4+) per nmol
of C2H4 produced.
For the natural dome (from August 1978 to
August 1979), the total fixation of 1.01 mg of N
per g of litter times the average litter fall of 390
g of organic matter per m2 per year yielded an
areal fixation rate of0.39 g ofN per m2 per year.
For the sewage dome, the total fixation of 0.208
g of N per g times the annual litter fall of 590 g
of organic matter per m2 per year yielded an
areal fixation rate of 0.12 g ofN per m2 per year.
Nitrogen exchange in decomposing cy-
press leaves. The original nitrogen concentra-
tion decreased significantly during initial decom-
position of the pond cypress leaves in litter bags
in the natural dome; after 1 month, the concen-
tration was only 81% of the initial value (Fig. 2).
This loss was later replenished in excess of the
original concentration. On the other hand, the
concentration in the pond cypress leaves decom-
posing in the sewage-enriched dome increased
immediately after placement in the dome.
CARY NATURAL DOME
FIG. 1. Seasonal variation in the nitrogen fixation rates determined for the surface layer ofpeat taken
from the natural dome and thesewagedomefrom 12 August 1978 to 12 August 1979. Eachpoint is the mean
ofthree to six replicates.
VOL. 41, 1981
DIERBERG AND BREZONIK
However, the absolute amount of nitrogen in
the leaf material remained relatively unchanged
(.90% of the original amount) in both domes
during the first year ofdecomposition (Table 1).
The lack of change resulted from a balance
between the increase in the nitrogen content of
the leaflitter and the loss oforganic matter from
the decomposing leaf litter (Table 1). Thus,
there was a net assimilation of nitrogen by de-
composers in relation to other dry weight com-
ponents that were lost from the litter. These
results indicate that nitrogen is not mobilized
and lost from recent litter fall but tends to be
retained inside the dome.
The percent contribution by heterotrophic
diazotrophs to the increase in nitrogen concen-
tration in decomposing litter ofthe naturaldome
was estimated as follows. After 4 weeks of de-
composition, the nitrogen content of the litter
had decreased to 6.58 mg ofN per g (81% of the
leached. (All concentrations here are expressed
cc - I0
FIG. 2. Nitrogen concentration inpond cypress leaves during decomposition in the standing waters ofthe
natural dome and the sewage dome. Each point is a mean offive values. The valuesplotted arepercentages
ofthe initial concentrations.
TABLE 1. Change in dry weight and carbon and nitrogen contents ofdecomposing leaf litter in two cypress
domes, 1978 to 1979
% of initial
aNumbers represent means of five bags.
bNumbers in parentheses represent ±1 standard error of the mean.
CARY NATURAL DOME
APPL. ENVIRON. MICROBIOL.
NITROGEN FIXATION AND DECAYING LEAVES
as dry weight.) After 4 months ofdecomposition,
the nitrogen content of the leaf litter increased
to 9.50 mg of N per g, or 117% of its original
level. Thus, during the 3 months when the nitro-
gen concentration of the litter was increasing, a
total of 2.92 mg ofN per g was added (or at least
conserved) compared with carbon and total or-
ganic matter. The total fixation during this pe-
riod was 0.408 mg of N per g (dry weight), as
determined by integrating the area under the
seasonal nitrogen fixation curve for the period
December 1978 to March 1979. Thus, 14% (0.408
mg/2.92 mg) ofthe nitrogen increment could be
due to nitrogen fixation.
Nitrogen concentrations in decomposing litter
from the sewage dome never decreased below
the original value (15.0 mg of N per g, dry
weight), because a constantly high concentration
of combined nitrogen (9.6 to 18.7 mg/liter) from
the sewage provided a more favorable milieu for
bacterial and fungal assimilation. This fact,
along with the low levels of nitrogenase activity
(Fig. 1), indicates that nitrogen fixation was a
negligible contributor of nitrogen to the decom-
posing litter of the sewage-enriched dome.
Increases in the concentration of an element
during decomposition can be explained either by
assimilation of the element from the environ-
ment by fungi and bacteria colonizing the litter
or by relative retention of the element in the
litter (compared with the bulk organic matter).
The extent to which these two processes occur
depends on the relative concentrations and
availability of the element in the litter and in
the immediate environment.
An increase in nitrogen concentration in de-
composing litter may also be due to nitrogen
fixation. In view of the nitrogen deficiency of
senescent leaves and the high ratio of carbon to
nitrogen in fresh litter (56.5 inthe natural dome),
nitrogen fixation could be expected to be an
important process in litter decomposition. Nu-
merous investigations have shown that hetero-
trophic nitrogenase activity (C2H2) can be stim-
ulated by adding simple carbon compounds (3,
9, 23, 24). Such stimulation is suggested by the
winterpeak ofnitrogenase activityinthe natural
dome, which followed the deposition of abscised
leaves from the two dominant trees (cypressand
blackgum) during October andNovember. Todd
et al. (22) also reported a winter peak in nitro-
genase activity (C2H2) for leaf litter in a decid-
Levels of combined inorganic nitrogenin the
surface water and sediment interstitial water of
the natural dome were relatively low (0.22 and
0.17 mg/liter, respectively). Nitrogen fixation
apparently supplements the normal absorption
ofcombined nitrogen by bacteria and fungi from
the aquatic environment as the organisms use
the carbon-rich litter substrate. While fixation
evidently does not provide a major part of the
nitrogen requirement of trees in the domes, the
fixation associated with decomposing leaf litter
does contribute nitrogen to an environment
short of available combined nitrogen. In addi-
tion, the 10- to 20-fold increase in nitrogenase
activity during the cold and dormant part of the
year occurs at a time when nitrogen released
from decomposing diazotrophs would be availa-
ble for spring growth of a new forest canopy.
Waughman and Bellamy (23) suggested that
fixation could contribute at least as much nitro-
gen as precipitation to some accreting peat for-
mations in Scotland. They found a relationship
between the hydrology ofmires (peat-producing
ecosystems) and nitrogenase activity; higher
acetylene reduction rates were associated with
peat from rheophilous mires, which receive sur-
face water inflows, than with peat from ombro-
philous mires, which receive only rainfall. This
finding was supported by Moore and Bellamy
(16), who summarized data on acetylene reduc-
tion in developing peat mires located in Europe
and Canada. For five sites with both ombrophil-
ous and rheophilous mires, no acetylene reduc-
tion was noted in any ombrophilous mires,
whereas acetylene reduction was observed at all
rheophilous mire types. The authors concluded
that the supply of nitrogen may be a factor
limiting the biological potential ofombrotrophic
mires. On the other hand, significant nitrogenase
activity was measured for surface peat from the
natural dome, which is an ombrophilous swamp.
The low acetylene reduction rates associated
with litter from the sewage dome are attributa-
ble to the repressive effects ofcombined nitrogen
from the sewage effluent on the synthesis of
nitrogenase. The peak in nitrogenase activity
beginning in May is not readily explained, but
may reflect an imbalanced N/P ratio. An exten-
sive bloom of Azolla caroliniana occurred in
the sewage dome 1 month before the measured
increase in acetylene reduction of the litter.
The absence of standing water in the natural
dome during November probably caused a lag
in nitrogenase activity of the freshly fallen leaf
litter. An earlier onset (Novemberor December)
of nitrogenase activity can be expected during
years when standing water is present duringthe
litter fall period.
Nitrogenase activitywas confined to the most
recent litter fall (i.e., the surface layer)incypress
domes, since only low ethylene productionrates
VOL. 41, 1981
1418 Download full-text
DIERBERG AND BREZONIK
were found below the surface in several cores.
On the other hand, acetylene reduction rates
were found to be uniform over the top 30 cm of
peat cores from British moors, although the
population ofpotential nitrogen fixers decreased
down the profile (14). Waughman and Bellamy
(23) sampled peat at a depth of 15 to 20 cm and
considered the C2H2 reduction rates measured
for these depths to be uniform up to the surface.
The results of the present study indicate that
rates of litter-associated nitrogen fixation and
mineralization are altered substantially when
natural cypress domes are used for disposal of
treated sewage. Nitrogen fixation rates associ-
ated with the decomposing leaf litter in the
sewage-enriched dome were much lower than
those in the natural dome, and fixation probably
did not contribute significantly to the nitrogen
economy of the sewage-enriched dome. These
findings, along with otherstudies on these domes
(5), indicate that nitrogen is conserved in cypress
domes. This retained nitrogen is available for
both internal cycling and storage in the woody
We acknowledge the assistance ofPete Straub in determin-
ing the nitrogen concentrations for the leaf litter.
This work was supported by National Science Foundation
Program of Research Applied to National Needs grant AEN
73-07823 A01 (formerly G 1-38721) and by Rockefeller Foun-
dation grant RF-73029 (H. T. Odum and K. C. Ewel, principal
1. Bremner, J. M. 1965. Total nitrogen, p. 1149-1178. In C.
A. Black (ed.), Methods of soil analysis, part IL Amer-
ican Society of Agronomy, Madison, Wis.
1977. Decomposition and nutrient ex-
change oflitter in an alluvial swamp forest. Ecology 58:
3. Brooks, R. IL, Jr., P. L. Brezonik, IL D. Putnam, and
A. Keirn. 1971. Nitrogen fixation in an estuarine
environment: the Waccasassa on the Florida GulfCoast.
Limnol. Oceanogr. 16:701-710.
4. de la Cruz, A. A., and B. C. Gabriel. 1974. Caloric,
elemental, and nutritive changes in decomposing Jun.
cU8 roemerianus leaves. Ecology 55:882-886.
5. Ewel, K. C., and H. T. Odum. 1978. Cypress swamps for
nutrient removal and wastewater recycling, p. 181-198.
In M. P. Wanielista and W. W. Eckenfelder, Jr. (ed.),
Advances in water and wastewater treatment: biological
nutrient removal. Ann Arbor Science Publishers, Inc.,
Ann Arbor, Mich.
6. Flett, R J., R D. Hamilton, and N. E. R. Campbell.
1976. Aquatic acetylene-reduction techniques: solutions
to several problems. Can. J. Microbiol. 22:43-51.
7. Gaudet, J. J. 1976. Nutrient relationships in the detritus
of a tropical swamp. Arch. Hydrobiol. 78:213-239.
8. Gotto, J. W., and B. F. Taylor. 1976. N2 fixation asso-
ciated with decaying leaves of the red mangrove (Rhi-
zophora mangle). Appl. Environ. Microbiol. 31:781-
9. Hanson, R. B. 1977. Comparison of nitrogen fixation
activity in tall and short Spartina alterniflora salt
marsh soils. Appl. Environ. Microbiol. 33:596-602.
10. Hardy, R. W. F., R. C. Burns, and R. D. Holsten. 1973.
Applications of the acetylene-ethylene assay for meas-
urement of nitrogen fixation. Soil Biol. Biochem. 5:47-
11. Howarth, R. W., and S. G. Fisher. 1976. Carbon, nitro-
gen, and phosphorus dynamics during leaf decay in
nutrient-enriched stream microecosystems. Freshwater
12. Jones, K. 1974. Nitrogen fixation in a salt marsh. J. Ecol.
13. Kauhik, N. K., and H. B. N. Hynes. 1971. The fate of
the dead leaves that fall into streams. Arch. Hydrobiol.
14. Martin, N. J., and A. J. Holding. 1978. Nutrient avail-
ability and other factors limiting microbial activity in
the blanket peat, p. 113-135. In 0. W. Heal and D. F.
Perkins (ed.), Production ecology of British moors and
montane grasslands. Springer-Verlag, New York.
15. Mathews, C. P., and A. Kowalczewskli. 1969. The
disappearance of leaf litter and its contribution to pro-
duction in the river Thames. J. Ecol. 57:543-552.
16. Moore, P. D., and D. J. Bellamy. 1974. Peatlands.
Springer-Verlag, New York.
17. Odum, E. P., and A. A. de la Cruz. 1967. Particulate
organic detritus in a Georgia salt marsh-estuarine eco-
system, p. 383-388. In G. H. Lauff (ed.) Estuaries.
American Association for the Advancement of Science
publication no. 83. Hom-Shafer, Baltimore.
18. Odum, IL T., K. C. Ewel, W. Mitach, and J. Ordway.
1975. Recycling treated sewage through cypress wet-
lands in Florida. Occasional publication no. 1. Center
for Wetlands, University of Florida, Gainesville.
19. Schell, D. M., and V. Alexander. 1970. Improved incu-
bation and gas sampling technique for nitrogen fixation
studies. Limnol. Oceanogr. 15:961-962.
20. Stewart, W. D. P., G. P. Fitzgerald, and R. EL Burris.
1967. In situ studies on N2 fixation using the acetylene
reduction technique. Proc. Natl. Acad. Sci. U.S.A. 58:
21. Thake, B., and P. R. Rawle. 1972. Non-biological pro-
duction ofethylene in the acetylene reduction assay for
nitrogenase. Arch. Mikrobiol. 85:39-43.
22. Todd, R. L, R. D. Meyer, and J. B. Waide. 1978.
Nitrogen fixation in a deciduous forest in the south-
eastern United States, p. 172-177. In U. Granhall (ed.),
Environmental role of nitrogen-fixing blue-green algae
and asymbiotic bacteria. Ecological bulletins (Stock-
holm), vol. 26. Swedish Natural Research Council,
23. Waughman, G. J., and D. J. Bellamy. 1972. Acetylene
reduction in surface peat. Oikos 23:253-258.
24. Zuberer, D. A., and W. S. Silver. 1978. Biological dini-
trogen fixation (acetylene reduction) associated with
Florida mangroves. Appl. Environ. Microbiol. 35:567-
APPL. ENVIRON. MICROBIOL.