Nitrogen Cycling in Coastal Marine Environments
Edited by T. H. Blackburn and J. S~rensen
(!;) 1988 SCOPE. Published by John Wiley & Sons Ltd
CHAPTER 5
Benthic Nitrogen Fixation
DOUGLAS G. CAPONE
5.1
INTRODUCTION
Interest in nitrogen fixation in coastal marine environments derives primarily
from the perception that nitrogen may be the factor limiting primary production.
Nearshore coastal and estuarine areas are characterized by high primary
production of plankton and rooted macrophytes (seagrasses and marsh grasses)
(Nixon and Pilson, 1983).Nitrogen, essential for plant growth, is generally found
in the shortest supply relative to estimated plant demands. It appears that the
sustained productivity of these systems results, in large part, from intense
recycling of entrained nitrogen. Nonetheless, sources of new nitrogen are
essential to offset losses of nitrogen removed or denitrified. In order to gain a
comprehensive understanding of the functioning of coastal ecosystems it is
important to assess all inputs of nutrients, including those from nitrogen fixation.
The demand for alternatives to costly, synthetic nitrogenous fertilizers in
agriculture has intensified efforts to exploit biological nitrogen fixation. Ironically, there is serious concern on a global scale over the rapid increase in
production of these synthetic fertilizers because of potential disruption of the
natural cycling of nitrogen (Delwiche, 1981).This has further prompted efforts to
evaluate more accurately rates of nitrogen fixation in nature (as well as other
nitrogen transformations) in order to place inputs derived from human sources in
perspective.
While there is little indication of a significant input of nitrogen by planktonic
diazotrophs in the nearshore region, nitrogen fixation is found in numerous and
varied benthic environments and appears to be a quantitatively important source
of new nitrogen in many of these systems. The purpose ofthis review is to provide
an overview of the considerable body of research which has accumulated on
nitrogen fixation in benthic marine environments.
5.2 N2 FIXATION AND NITROGENASE
Basic aspects of the biochemistry, enzymology, physiology, genetics and ecology
ofN2 fixation havebeensuccinctlysummarizedby Postgate(1982),and readers
86
Nitrogen Cycling in Coastal Marine Environments
are referred there for a more thorough discussion ofNz fixation and nitrogenase.
In brief, biological nitrogen fixation is the reduction of dinitrogen (Nz) to NH3'
The reaction can be represented as:
Nz + 8H + + 8e -
~
2 NH 3 + Hz
I
. (amino acids)
(1)
The ammonia formed is subsequently incorporated into cellular material. The
process is catalyzed by the molybdo-iron enzyme, nitrogenase, which occurs only
in a disparate grouping of prokaryotic organisms.
There are a number of common properties of the nitrogenases isolated from
bacteria. Nitrogenase is an enzyme complex consisting of two major proteins,
each of several subunits. The combination of a Mo-Fe protein, or 'dinitrogenase'
(the likely site of Nz reduction) and a Fe protein or 'dinitrogenase reductase' is
required to form a functional nitrogenase system. Components from diverse
bacteria have been combined to produce active preparations. The homology
among the nitrogenase enzymes of the diverse groups of bacteria that possess Nz
fixing ability is striking, and has led to considerable speculation on the means of
dissemination of nitrogenase among these groups.
A large metabolic cost is associated with this reaction and is estimated to be the
equivalent of about 15 ATP per N z reduced (postgate, 1982). In the various
diazotrophs known, this metabolic cost is met through phototrophic or
chemotrophic pathways. Hydrogen evolution is apparently a natural sideproduct of the process. All enzyme preparations studied to date, including those
isolated from strictly aerobic bacteria, are inhibited by Oz. However, the degree
of °z sensitivity of nitrogenase is less in aerobic bacteria than in anaerobes.
Ammonium appears to regulate the synthesis of nitrogenase, although the details
of regulation vary among the diazotrophs. For a number of Nz fixing systems,
regulation of nitrogenase appears to be intimately related to regulation of the
next enzyme in the assimilatory pathway, glutamine synthetase (Streicher et ai.,
1974; Dixon, 1984). The nitrogenases of all systems studied show a very low
specificity for the natural substrate and are capable of reducing a broad range of
analogous molecules. Indeed, this property has been exploited in developing the
CzHz reduction assay, the routine means of determining nitrogenase activity (see
Section 5.4).
Recently, the presence of a non-molybdenum dependent nitrogenase has been
confirmed in Azotobacter chroococcum (McKerra et ai., 1970; Robson et ai.,
1986). This enzyme system, which also reduces CzHz, is induced during Mo
starvation and has a vanadium cofactor.
5.3 Nz-FIXING BACTERIA
N z-fixingbacteria were first cultured from seawater and marine muds by Benecke
and Keutner (1903). Waksman et ai. (1933), summarizing the earlier studies of
Benthic Nitrogen Fixation
87
others' as well as their own work, concluded that Azotobacter was common in the
water column, at the sediment surface and on many macro algae, while
Clostridium appeared to be a ubiquitous inhabitant of the marine bottom. They
directly demonstrated nitrogen fixation in their enrichment and pure cultures by
the crude methods available at the time (increases in cellular N during growth on
'N-free' media), properly recognizing the limitations of their procedures as well as
the inappropriateness of pure culture studies to understanding in situ activites. In
their words:
However, in view of the fact that nitrogen-fixing organisms do not live in the sea in
the form of pure cultures, but are there as mixed populations, a knowledge of their
ability to fix nitrogen in such a mixture is more important than in pure culture. It
still remains to be determined, however, to what extent this process actually takes
place in the sea.
They thus set the stage for the ecological studies which were initiated about
30 years later.
Nonetheless, pure culture studies are conspicuous in the literature and are of
inestimable value in ascribing the process to specifictypes of bacteria. In addition,
they provide experimental organisms for defining the physiological and nutritionallimits of the activity, and serve for comparative genetic studies. A broad
range of marine diazo trophic bacteria have been cultured and the presence of
nitrogenase has been confirmed using either the uptake of 15N2 or the reduction
of C2H2 (see Section 5.4).
A brief summary of representative genera of heterotrophic diazotrophs
enriched or isolated from marine environments is presented in Table 5.1 and a
similar compilation for photoautotrophic forms appears in Table 5.2. Seven of
the 28 genera of heterotrophic diazotrophs listed by Postgate (1982) have
confirmed marine representatives. The diazotrophic Campylobacter noted by
Postgate (1982)was isolated from Spartina by McClung and Patriquin (1980)and
is the only known N2 fixer in this genus.
Two of the genera listed in Table 5.1 are recent isolates and were not included
in Postgate's (1982) summary. The marine Vibrio species isolated by Guerinot
and Patrinquin (1981a,b); and Guerinot et al. (1982) are unique among the
Vibrionaceae in possessing nitrogenase. Nelson et al. (1982) were the first to
confirm nitrogenase activity in marine isolates of the genus Beggiatoa. Waterbury
et al. (1983) have isolated a N2-fixing, cellulose-digesting aerobic chemoheterotroph from the gland of Deshayes in shipworms (teredinids) which does not fit in
any known bacterial genus. Guerinot and Colwell (1985) have also noted an
apparently novel genus of diazotrophic marine bacteria, as well as the presence of
nitrogenase activity in isolates of Oceanospirillum(Azospirillum?).
Several bacterial genera which have diazotrophic members are known to occur
in marine environments (Table 5.3)and it remains to examine marine isolates for
nitrogenase activity. For instance, the widespread distribution of methane-
00
00
Table 5.1. Heterotrophic N2-fixing bacteria enriched or isolated from marine environments
Relation Genus
to O2
1. Aerobes
Azotobacter spp.
2. Microaerophiles
Azospirillum spp.
Campylobacter spp.
Beggiatoa spp.
(?)
Enumeration
or isolation
Habitat"
References
E
E
I
ElI
Ell
Ell
Black Sea sediments
Seagrass sediments
Macroalga Codium
Intertidal sediments
Estuarine sediments
Saltmarsh sediments
Pshenin (1963)
Patriquin and Knowles (1972)
Head and Carpenter (1975)
Herbert (1975)
Jones (1982)
Dicker and Smith (1980b)
I(?)
I
I
I
I
I
Spartina roots
Zostera roots
Seawater
Spartina roots
Sediment surface
Shipworm
Patriquin (1978b)
Budin (1981)
Guerinot and Colwell (1985)
McClung and Patriquin (1980)
Nelson et al. (1982)
Waterbury et al. (1983)
~
..,
0
<Q
'"
;:
()
'..:
k
S.
<Q
s.
()
0
$:>
'"
[
~
$:>
..,
S.
'"
t'1
;:
'"
0::t.
;:
S!
'"
i::.
'"
t:I::i
II>
;:s
~
;:;.
~
3. Facultative anaerobes
Enterobacter spp.
Klebsiella spp.
Vibrio spp.
4. Strict anaerobes
Desulfovibrio spp.
Clostridium spp.
I
Ell
Ell
Ell
I
I
I
I
I
swlbeach sediment
Intertidal sediments
Estuarine sediments
Halodule roots
swlbeach sediment
Mangrove bark
Sea urchin
Seawater
Mangrove bark
Werner et al. (1974)
Herbert (1975)
Jones (1982)
Smith and Hayasaka (1982b)
Werner et al. (1974)
Uchino et al. (1984)
Guerinot et al. (1982)
Guerinot and Colwell (1985)
Uchino et al. (1984)
E
Ell
Ell
Ell
E
E
Ell
Ell
I
E
Seagrass sediments
Intertidal sediments
Saltmarsh sediments
Estuarine sediments
Estuarine sediments
Seagrass sediments
Intertidal sediments
Saltmarsh sediments
Estuarine sediments
Estuarine sediments
Patriquin and Knowles (1972)
Herbert (1975)
Dicker and Smith (1981)
Jones (1982)
Blake et al. (1982)
Patriquin and Knowles (1972)
Herbert (1975)
Dicker and Smith (1981)
Jones (1982)
Blake et al. (1982)
...
<::>
"'"
II>
;:s
.."
>;'
::.
o'
;:s
'sw = seawater
00
\0
90
Nitrogen Cycling in Coastal Marine Environments
Table 5.2. Phototrophic N2-fixing bacteria isolated from marine environments
1. Cyanobacteria
A. Chroococcacean
Synechococcus sp.
Gloeocapsa sp.
B. Pleurocapsalean
Dermocarpa sp.
X enococcus sp.
Myxosarcina sp.
Pleurocapsa sp.
C. Oscillatorian
LPpa group
Oscillatoria sp.
Microcoleus sp.
Phormidium sp.
D. Nostacalean
Anabaena sp.
Calothrix sp.
N odularia sp.
N ostoc sp.
2. Prochlorales
Prochloronb
3. Anoxyphotobacteria
A. Rhodospirillaceae
Rhodopseudomonas sp.
B. Chromatiaceae
Thiocapsa sp.
References
Habitat
Group and genus
Snail shell, intertidal
Tropical sediments
Microbial mat
Rippka and Waterburry (1977)
Duerr (1981)
Stal and Krumbein (1985)
Marine aquarium
Marine aquarium
Snail shell, intertidal
Rock chip
Rippka
Rippka
Rippka
Rippka
Snail shell, intertidal
Intertidal mat
Marsh mat
Microbial mat
Rippka and Waterbury (1977)
Stal and Krumbein (1981)
Pearson et al. (1979)
Stal and Krumbein (1985)
Algal mat
Algal mats
Microbial mat
Microbial mat
Microbial mat
Stacey et al. (1977)
Gotto et al. (1979a)
Stal and Krumbein (1985)
Stal and Krumbein (1985)
Stal and Krumbein (1985)
Ascidians
Paerl (1984)
Harbour muds
?
Wynn-Williams and Rhodes
(1974)
Kelley et al. (1979)
'Anaerobic habitat'
Jouanneau et al. (1980)
and
and
and
and
Waterbury
Waterbury
Waterbury
Waterbury
(1977)
(1977)
(1977)
(1977)
, LyngbyajPlectonemajPhormidium
group.
b Presumptive fixer, only active in association.
oxidizing bacteria has been recently noted (Sieburth, personal communication;
Sieburth et al., 1987). The presence of nitrogenase in various methylotrophic
bacteria is well known, and their importance as N 2 fixers in some aquatic
environments has been demonstrated (Rudd et al., 1976). A variety of chemoautotrophic bacteria are recognized N 2 fixers (e.g. Thiobacillus, hydrogenoxidizing bacteria). Autotrophic sulfide- and hydrogen-oxidizing bacteria are
also thought to be part of the natural marine micro biota (Schlegel, 1975;Kepkay
et al., 1979). Most recently, two methanogenic bacteria have been confirmed
as diazotrophs (Murray and Zinder, 1984; Belay et al., 1984). Methanogenic
bacteria can be an important part of the anaerobic flora in marine sediments
(Oremland, 1987).
91
Benthic Nitrogen Fixation
Table 5.3. Heterotrophic and chemautotrophic genera of diazotrophic
(Postgate, 1982) for which marine diazotrophs have not been observed
Relation
to Oz
1.
2.
3.
4.
Genus
Aerobes
Azomonas
Azotococcus
Beijerinckia
Derxia
Xanthobacter
Microaerophiles
Aquaspirillum
Arthobacter
Frankia
Methylosinus
M ethylocystis
Methylomonas
Methylobacter
Thiobacillus
Rhizobium
Alcaligenes
Facultative anaerobes
Erwinia
Citrobacter
Escherichia
Bacillus
Strict anaerobes
Desulfotomaculum
Methanosarcina
Methanococcus
Comments
( = Azotomonas
bacteria
References
?)
(Hz oxidizers)
Marine species
Sieburth (1979)
Marine species
Sieburth (personal communication)
Marine species
Sieburth (1979)
Marine species
Sieburth (1979)
Marine species
Sieburth (1979)
Marine species
Marine species
Sieburth (1979)
Sieburth (personal communication)
All major groups of cyanobacteria have diazo trophic representatives isolated
from marine environments, including the coccoid and non-heterocystous
filamentous forms (Table 5.2). Of the 20 genera of cyanobacteria with known
diazo trophic members mentioned by Postgate (1982),thirteen marine representatives have been confirmed to have nitrogenase. The Nz-fixing Anabaena strains
reported by Stacey et al. (1977)and Gotto et al. (1979a)have amongst the highest
growth rates reported for heterocystous cyanobacteria growing under Nz. A
recent observation is that of Paerl (1984), who attributes nitrogenase activity
associated with some didemnid tropical acidians to Prochloron symbionts. The
activity only occurs in the intact association, and does not persist when the
symbionts are separated from the host. This is an exciting development from both
an ecological and an evolutionary viewpoint, and deserves further effQrts at
confirming the presence of nitrogenase in Prochloron.
92
Nitrogen Cycling in Coastal Marine Environments
All three major groups of photosynthetic bacteria (anoxyphotobacteria) have
genera which possess nitrogenase activity. While diazotrophic marine representatives of the purple non-sulfur (Rhodospirillaceae) and sulfur (Chromatiaceae)
bacteria have been obtained in pure culture, I found no indication for the
enrichment or isolation of Nrfixing marine forms of the green sulfur bacteria.
5.4 METHODOLOGY
Initial indications ofthe presence of nitrogen-fixing bacteria in the sea came from
isolation and enrichment studies (see above). Rudimentary attempts at estimating
rates of Nz fixation were made by determining increases in cellular nitrogen in
presumptive diazotrophs grown on N-free media (Waksman et al., 1933;
Table 5.4. Summary of methods of determining Nz fixation
Method
1. Qualitative
(a) Enrichment,
N-free media,
MPM
(b) Enrichment,
confirm CzHz
reduction
(c) Isolation of
diazotrophs
2. Quantitative
(a) Increase in total
N (Kjeldahl)
(b) Manometry
Comment
Relative
sensitivity'
Enumeration
References
(See Tables 5.1-5.3)
(See Tables 5.1-5.3)
(See Tables 5.1-5.3)
Imprecise
Imprecise
(c) 15Nz reduction
(d) CzHz reduction
Precise, tedious
Simple, precise
(e) 13Nz reduction
(f) 15N-fertilizer
isotope dilution
(g) Membrane-leak
mass spectrometry
(h) Natural
abundance 15N
Direct, exotic
Untested, marine
environment
, From Schell (1978); Cooper et at. (1985).
1
Burns and Hardy
(1975)
Burns and Hardy
(1975)
103
Burris (1974)
106
Burns and Hardy
(1975); Burris (1974)
1010 - 1012 Cooper et al. (1985)
?
Boddey et al. (1983)
?
?
Jensen and Cox
(1983)
Amarger et al.
(1979)
93
Benthic Nitrogen Fixation
Table 5.4).The insensitivity of such an approach, the ability of some organisms to
grow efficiently on traces of combined nitrogen (Postgate, 1982)and the use of
pure cultures made the ecological interpretation of such data difficult. Direct
evaluation of the quantitative importance of in situ N2 fixation awaited the
development of appropriate methods.
The first quantitative estimates ofN 2 fixation in marine systems were made via
15N2uptake studies. Dugdale et ai. (1961)and Stewart (1965,1967) pioneered the
use of this isotope for N 2 fixation studies in pelagic and benthic environments,
respectively, adapting it from the biochemical tracer methods of Burris and
Wilson (1957).After exposure of a sample of interest to N2 highly enriched in 15N,
the increase in the ratio of 15Nto 14Nin the biological material, compared to the
naturally occurring ratio ( '" 0.36%),provides a measure of N 2 fixation. While
15N2 methods do provide unequivocal and direct qualitative evidence for this
process, quantitative interpretation of the data in organic-rich sedimentary
systems may provide particular problems (see below). Furthermore, the procedure is not amenable to large-scale field programs. The mass spectrographic
method is inherently limited in its sensitivity, speed and sample throughput. This
has precluded its wide-scale application. The alternative use of optical emission
spectroscopy to quantify the isotopic ratio of 14N to 15N (Meyer et ai., 1974)
provides no particular remedy other than the lower cost of the analytical
instrument.
The development of the C2H2 reduction assay (Stewart et ai., 1967; Hardy
et ai., 1968) revolutionized the study of N2 fixation in nature by providing a
sensitive and simple procedure for determining nitrogenase activity. There has
been a steady increase in reports on marine nitrogen fixation from 1972 onward
(Capone and Carpenter, 1982b), due mainly to the availability of the C2H2
reduction method. The procedure relies on the low specificity of nitrogenase for
its natural substrate and its capacity to reduce other triply bonded small
molecules. In particular, C2H2 is reduced to C2H4 by nitrogenase:
C2H2 + 2H+ + 2e- -+C2H4
(2)
In fact, nitrogenase reduces acetylene in preference to its natural substrate, N 2'
The ethylene formed after addition of the alternate substrate is easily quantified
by flame ionization gas chromatography.
The C2H2 reduction method is not without its problems, however, particularly with respect to its application for quantitative estimates of N2
fixation in situ. The advantages, limitations and judicious use of the assay have
been recently reviewed and discussed by Taylor (1983), Payne (1984) and
Oremland and Capone (1987).A primary consideration is the fact that C2H2, at
the concentrations required in the assay, is inhibitory to a broad range of both
diazo trophic and non-diazo trophic microorganisms. These include methanogens
(Oremland and Taylor, 1975),methane oxidizers (Dalton and Whittenbury, 1976;
DeBont, 1976;DeBont and Mulder, 1974),nitrifiers(Hynesand Knowles,1978),
94
Nitrogen Cycling in Coastal Marine Environments
denitrifiers (Yoshinari and Knowles, 1976)and sulfate reducers (Payne, 1984).By
its very nature the assay substitutes the reduction of CzHz for N z fixation, and
should thereby induce nitrogen starvation in those organisms dependent upon
Nz fixation for nitrogen (Brouzes and Knowles, 1971).Indeed, David and Fay
(1977) have noted accelerating nitrogenase activity, possibly from increased
synthesis of enzyme, after extended exposure to CzHz in cyanobacteria. In
systems characterized by complex nutritional interrelationships among the
micro biota, such as organic-rich marine sediments, differential inhibition of the
microbiota may compromise the accuracy of estimates of Nz fixation derived
from CzHz reduction. The nature and susceptibility of the microbial flora to
CzHz inhibition, the nutritional status ofthe sample and, in particular, the length
of exposure to CzHz, will all contribute to departures from accurate estimates of
in situ Nz fixation. Because of the extreme sensitivity of the flame ionization
detector for ethylene, only brief exposure periods (seconds to minutes) to CzHz
are required before rates of CZH4production can be measured, and these shortterm rates probably best represent in situ activities. Estimates of Nz fixation
derived from rates of CzHz reduction after extended lag periods cannot be
interpreted in quantitative terms (Patriquin, 1982).A further point of concern is
the recent observation of Hyman and Arp (1987) of various contaminants in
CzHz preparation which can either stimulate or inhibit nitrogenase activity.
Ethylene production can occur naturally, and controls used in CzHz reduction
studies often include an acetylene-freeflask intended to correct for endogenous
CZH4 production. Witty (1979), working in a terrestrial soil, and Howarth
(personal communication), working in a saltmarsh peat, observed that in the
presence of 14CzHz at assay concentrations, a significant accumulation of
unlabeled CZH4 occurred, presumably not from CzHz reduction. Hydrocarbonoxidizing bacteria are known to be quite sensitive to CzHz (DeBontand Mulder,
1974). Apparently, in some aerobic soil and sediment systems, low ambient
concentrations of ethylene belie a dynamic ethylene cycle in which excess
consumption of ethylene prevents its accumulation. The introduction of CzHz
into such a system blocks CZH4consumption and results in its build-up. The use
of the CzHz-free control does not reveal this phenomenon. Witty (1979)
recommends use of 14CzHz to uncover the potential for this artifact. However,
incubation of samples with CZH4 in the absence of CzHz should also provide
evidence for a high natural rate of CZH4 oxidation (Van Berkum and Sloger,
1979). Van Berkum and Sloger (1979) did not observe this phenomenon in
saltmarsh grass (Spartina) systems incubated aerobically. Although CzHz can be
oxidized both aerobically (1976; DeBont and Mulder, 1974) and anaerobically
(Culbertson et al., 1981), there is no evidence for oxidation of CZH4 under
anaerobic conditions (Figure 5.1; C. Culbertson, 1983, M.s. thesis, S.F. State
University; Oremland, personal communication).
The rate of ethylene formation is related to N z fixation by consideration of the
reducing equivalents required for acetylene reduction to ethylene ( = 2)compared
95
Benthic Nitrogen Fixation
600
400
I
C2H2 (gas phase)
I-....\
(a)
\
300
(")
\
\
'"°
\
\
\
\
I
200
.
\
i roo
N
\
\
.....
CO2 (gas phase)
\
-.
\
400r
1:
3
0
100
\
0
'"
""
N
U
0
5
10
2000~_.-~,~
~
150
200
~-----
1600L C2H4 (gas phase)
--j150
(b)
~
.2
"-
(")
1200
100
.....
0
E
:l
1:
3
0
800
CO2 (gas
phase)
50
I"N 400
:::::
-.
0"
'"
""
U
0
",°
5
10
15
0
Days
Figure 5.1. Inability of acetylene-utilizing enrichment cultures
to grow on ethylene. (a) Acetylene control (total C2H2 added,
800 J.lmol).(b) Flask containing C2H4 as a growth substrate (total
C2H4 added, 3500J.lmol).Points are single flask determinations.
(From Culbertson (1983), M. S. thesis, S. F. State University.)
to those needed for N 2fixation ( = 6)or a theoreticalratio of3 molesofacetylene
reduced per mole of N2 fixed. Physiological, toxicological and enzymological
factors may result in deparatures from ideal stoichiometry. The ratio is probably
higher in most systems since reducing equivalents are also shunted to H2
production under N2 fixing conditions, but not during C2Hz reduction (compare
equations 1 and 2) (Postgate, 1982).In practice the method should be calibrated
against a direct method (e.g. 15N2 fixation) for each particular system in order to
substantiate its quantitative usefulness.
While the CzHz reduction method has found wide application in marine
systems, there have been relatively few comparisons with direct measures of
nitrogen fixation, particularly for sedimentary systems (Capone, 1983b). Sediments presenta particularly difficult subjectfor such comparisonsbecauseof
96
Nitrogen Cycling in Coastal Marine Environments
large pools of adsorbed ammonium (Rosenfeld, 1979; Mackin and Aller, 1984)
and detrital organic nitrogen (Rice, 1982) (see below). Some workers have
obtained reasonable agreement between 15N2fixation and C2H2 reduction in
benthic marine systems such as algal mats (Potts et al., 1978) seagrass roots
(Patriquin and Knowles, 1972; Capone and Budin, 1982) and decomposing
mangrove leaves (Hicks and Silvester, 1985).On the other hand, Seitzinger and
Garber (1987) have been unable to reconcile 15N enrichment in sediments after
exposure to 15N2, to rates of C2H2 reduction. They report ratios of C2H2
reduction to N2 fixation from 10:1 to 100:1, and suggest that observed C2H2
reduction in this environment may not be assessing nitrogenase activity, at least
in any quantitative sense.
Various independent factors do strongly indicate that C2H2 reduction in
sedimentary systems is a result of nitrogenase activity. The oxygen sensitivity and
ability of NH: to inhibit C2H2 reduction in various sediment systems (see
Section 5.5) are consistent with this conclusion. The recent observation that
removal of interstitial NH: by perfusion (Capone and Carpenter, 1982a) or the
addition of methionine sulfoximine (a derepressant of nitrogenase synthesis)
rapidly stimulate C2H2 reduction in saltmarsh and seagrass sediments (Capone,
1984; Yoch and Whiting, 1986; Section 5.5) are further evidence that observed
acetylene reduction derives directly from nitrogenase activity.
In complex sample matrices such as sediments, quantitative interpretation of
15N2uptake data and comparisons with C2H2 reduction are problematic. The
calculation of nitrogen fixation rates from 15N2 reduction depends on the
accurate estimate of the 15Nto 14N ratio of the substrate N2 and of the sample of
interest after an exposure period to 15N-enriched N 2' and of the 'organic'
nitrogen content of the sample.
APE,ample x N content,ample = N fixed
N2
where APE = atom% excess 15N in nitrogen.
This is a straightforward procedure for monospecific or relatively simple
diazotrophic systems in nature (e.g.root nodules, cyanobacterial mats) which can
be cleanly separated and analyzed. In soils and sediments, however, the
diazotrophic flora is part of a larger microbial population and is only a minuscule
fraction of the particulate nitrogen pool. Garber (1982) estimates bacterial
biomass in Narragansett Bay sediments to be about 0.01% of total combined
nitrogen in the sediments. The largest pool of nitrogen in this system was detrital
(non-living)organic nitrogen ('" 98.7%).Much of the organic nitrogen in sediments
is recalcitrant geopolymer, the products ofhumification (Rice, 1982).Inclusion of
this pool in extracted or digested samples for isotope ratio analysis dilutes the
apparent level of enrichment and radically decreases the sensitivity of the assay.
On the other hand, there are no current procedures for fractionation and
Benthic Nitrogen Fixation
97
isolation of the relevant 'living' nitrogen pool. Protein is associated and
complexed with detritus as well as with living biomass (Mayer et ai., 1986;Rice,
1982). Recent improvements in amino acid separation and analysis (e.g.
glutamate and glutamine pools) may lend themselves to a solution to this
problem, although the ability to obtain sufficient sample for mass spectrometry
may need to be overcome.
With presently used procedures, 15Nz fixation is probably incapable of
providing an appropriate means of calibrating the CzHz reduction method in
marine sediments. Table 5.5presents a theoretical consideration of apparent lsN
enrichment within various pools of nitrogen for a given enrichment within the
diazotrophic bacterial pool. For precise quantitation of enrichment by mass
spectrometry, a minimum of 0.003% 15N excess is required (Burris, 1974).Using
this criterion, and assuming linear uptake, sufficient enrichment could be
observed in the 'living nitrogen pool' after 1h if this pool could be cleanly
separated. It would, however, require a 20 h incubation to detect even this
minimum level in the particulate nitrogen pool at a rate of Nz fixation
comparable to the highest rates of CzHz reduction reported when converted to
N-equivalents(seeTable 5.10).Thismaybe considereda 'bestcase'scenariowith
respect to the assumed size (10% of bacterial population) and activity
(3.1ng N cm - 3 h - 1) of the diazo trophic bacterial population, and hence the rate
of organic nitrogen enrichment. For comparison, using the high and low rates of
CzHz reduction (0.5and 0.02nmol g dry sed - 1 h - 1)observed in shallow subtidal
non-rhizosphere temperate estuarine sediments (Capone, 1983b)and assuming a
sediment density of 1.5gjcm3 and an CzHz to Nz ratio of 4, it is possible to
calculate estimated N z fixation rates of 5.3 and 0.1ng N cm - 3 h - 1. To further
illustrate the point, Jones (1974)reported an observed enrichment of only 0.455
APE in a Spartina soil after 7 days exposure to 90 atom% excess 1sNz. There is
thus a basic incompatibility in making comparisons between CzHz reduction
and 1sNz reduction in sediments, since the former is most appropriately applied
in brief assay while 1sNz reduction require long-term incubation to provide
sufficient enrichment to be detected (Buresh et ai., 1980).
Other approaches may be available to determine the relationship between
CzHz reduction and Nz fixation in sediments. 13Nzmethods have been used in
nitrogen fixation studies and may provide a more appropriate direct means for
'calibrating' CzHz reduction methods (Cooper et ai., 1985).This method is
extremely sensitive, with detection limits of the order of less than 5 fmol of
product (Table 5.4)(Cooper et ai., 1985).Schell (1978)estimates 13Nmethods to
be about 108times more sensitive than 1sN. Alternately, determination of Hz flux
in the presence and absence of CzHz may allow an estimate of the departure
from an ideal 3: 1 stoichiometry (Saito et ai., 1980). However, fermentative
production and anaerobic oxidation of Hz in sediments make this impractical.
The rate ofNz flux into the liquid phase as a function of nitrogen consumption
by nitrogen fixation and simultaneousdeterminationof Hz flux associatedwith
Table 5.5. Hypothetical estimate of enrichment in 15N in various nitrogen pools for a given rate of N 2
N pool
Diazotrophic
bacteria
Total bacteria
Total living Na
Particulate Na
fixation
N-content
(g/cm3)
Percentage 15N
APE (h)
N 2 fixation
(ng cm - 3 h - I)
N turnover
(percentage cell N/h)
0.7
7.0
20.0
1600
0.720
0.396
0.3723
0.3602
0.360
0.036
0.0126
0.0002
3.1
3.1
3.1
?
0.45
a From Garber (1982), cited in Nixon and Pilson (1983).
Data for meiofaunal and macrofaunal N (13 Jlg/cm3) and total detrital (particulate) N were taken from Garber (1982). The estimate by Garber (1982) of
0.20 JIg/em 3 for bacterial biomass is inconsistent with other literature values. Hence a value for bacterial biomass of 50% of combined macro- and
microfaunal biomass as bacterial biomass, derived from Gerlach (1978) and Meyer-Reil (l983)was used instead, yielding a total living biomass of20 Jlg/em3.
Diazotrophic bacterial biomass was assumed to be 10% of total bacterial biomass. An 80% enrichment of I'N2 and an increase of 0.360 atom% of nitrogen
each hour in the biomass of diazo trophic bacteria was assumed. For the present argument this translates to a rate ofN2 fixation of3.1 ngcm -3 h -I, whieh is
comparable to the highest recorded values for unvegetated, shallow subtidal sediments.
Benthic Nitrogen Fixation
99
cyanobacteria in steady state have been measured by membrane leak mass
spectrometry (Jensen et aI., 1981;Jensen and Cox, 1983).The method was in good
agreement with other estimates ofN2 fixation, but may only be useful in systems
with relatively high N2 consumption and without denitrification. Incidentally,
Jensen and Cox (1983) also showed a good correspondence between the
departure from a theoretical 3:1 ratio of C2H2 reduced to N2 fixed and the
inhibition of nitrogenase-dependent H2 production under C2Hrreducing
conditions.
Another application of 15N2 fixation methodology, the N-fertilizer isotope
dilution method (Boddey et aI., 1983; Lethbridge and Davidson, 1983; Witty,
1983), has been used recently in soils. After addition of 15N-labeled fertilizer of
known enrichment to an experimental crop plot, the concentration of available
nitrogen and the ratio of 15Nto 14Nin this pool are monitored, a method directly
analogous to the NH: turnover methods of Blackburn (1979). The technique
requires an appropriate 'control' plot of a non-nitrogen-fixing crop with similar
plant root uptake characteristics and with directly comparable soil N pools
(Witty, 1983). The method is suitable for estimation of the nitrogen fixation
contribution to plant nutritional needs over long (seasonal) time scales and where
appropriate controls can be obtained (Witty, 1983).The potential application for
marine studies is improbable.
Finally, the natural abundance of 15N to 14Nin samples has been suggested as
a qualitative method of screening for possible diazotrophic associations because
of the generally lower 15N:14N ratio found in such association (Delwiche et at.,
1979).Several workers (Amarger et at., 1979;Kohl et at., 1979)have attempted to
make quantitative estimates of contributions from nitrogen fixation to plant
nitrogen requirements. However, insufficient information on the mechanisms of
fractionation (Delwiche et aI., 1979) and inconsistencies in isotope abundance
trends in nitrogen-fixing plants (Turner and Bergersen, 1983;Turner et at., 1983)
relegates to the future an evaluation of the usefulness of such an approach.
5.5 PHYSIOLOGICAL ECOLOGY: REGULATING FACTORS
A broad range of environmental and biochemical factors can affect the nature
and extent of N 2 fixation within a particular environment or system. Light,
temperature, pH, salinity, °2' micro nutrients, inorganic nitrogen and organic
substrate availability have all been shown to modulate nitrogenase activity in
specific situations. None of these factors has been extensively or systematically
examined for its role in controlling nitrogenase activity in benthic environments.
Indeed, some may be irrelevant in the context of sedimentary N2 fixation.
Nonetheless, a number of studies have made cursory examinations of one or more
of these factors within particular systems. The present discussion will focus on the
better-studied factors controlling heterotrophic nitrogen fixation in the sedi-
100
Nitrogen Cycling in Coastal Marine Environments
ments, namely the effects of O2, inorganic nitrogen and organic substrate on
activities of natural populations.
In shallow benthic environments, physicochemical parameters can exhibit
extreme variability. Gradients of gases and inorganic constituents in the sediment
column are well documented (Billen, 1982; Revsbech et al., 1980). Light is
certainly a factor in photoautotrophic nitrogenase activity at the surface of
shallow sediments. Numerous studies have observed light-stimulated or lightdependent activity (Whitton and Potts, 1982).Similarly, temperature fluctuations
affect microbial populations and metabolism in general, including nitrogenase
activity. Such fluctuations are likely to be most pronounced at or near the
interface, and become damped with depth into the sediments. Nonetheless,
seasonal patterns of activity (e.g. Marsho et al., 1975;Hanson, 1977a; Patriquin
and McClung, 1978; Capone and Taylor, 1980a; Dicker and Smith, 1980c;
Capone, 1982;Jones, 1982)and the short-term effectof temperature variation (e.g.
Capone and Taylor, 1980a; Jones, 1982;Smith and Hayasaka, 1982a,b) are well
documented for N 2 fixation in sedimentary systems of both subtropical and
temperate climes.
Only minor variations in pH occur in the well-buffered medium of seawater;
hence, few studies have considered pH as an important regulatory factor in situ.
Similarly, in most marine systems (from a biological standpoint) salinity
fluctuations are minor and generally not considered to affectnitrogenase activity.
However, in estuarine areas salinity may vary from 0%0to> 30%0over short
temporal and spatial scales.Several studies have examined the effect of salinity on
nitrogenase activity of isolates (e.g. Herbert, 1975;Dicker and Smith, 1981)and
natural populations (Ubben and Hanson, 1980).Not surprisingly, in some strains
nitrogenase activity appears to be salt-dependent, in others it displays broad salt
tolerance and yet in others nitrogenase activity is inhibited at high salinities
(Herbert, 1975).
Nitrogenase has co-factor requirements for molybdenum and iron. In some
soil systems Mo has been found to be in short enough supply to limit N2 fixation.
It has recently been suggested that N 2 fixation in the water column of marine
ecosystems may be limited by availability of either iron (Reuter, 1984) or
molybdenum (Howarth et al., 1984;Howarth and Cole, 1985).Howarth and Cole
(1985) have proposed that the high concentrations of sulfate ion in seawater
interfere with MoO~ - acquisition by pelagic diazotrophs. Such limitation is
unlikely in sediments because of the relatively high concentrations of these
me-tals(Malcolm, 1985).The possibility of vanadium dependent nitrogenases in
Mo limited marine environments deserves some consideration.
5.5.1
Oxygen
Oxygen is a major determinant of nitrogenase activity at both an enzymological and ecological level. In shallow coastal sediments, oxygen decreases rapidly
101
Benthic Nitrogen Fixation
with depth, and is often undetectable within millimeters of the sediment-water
interface. Despite the fact that nitrogenase is an extremely °2-sensitive enzyme,
nitrogenase activity occurs in fully aerobic through anoxic zones. Marine isolates
have diverse physiologies with respect to oxygen, ranging from strict aerobes to
strict anaerobes. Enrichment studies have noted marked variability in the
proportion of aerobic and anaerobic N 2 fixers in marine sediments.Herbert
(1975), Jones (1982) and Blake et al. (1982) all found a preponderance of
anaerobic and facultative diazotrophs in the sediments they examined, while
Dicker and Smith (1980b) also recorded large populations of aerobic as well as
anaerobic N2 fixers in saltmarsh sediments.
Surprisingly few studies have considered the relationship of natural populations of diazotrophs to °2. The responses which have been noted are as varied as
the samples studied (Table 5.6). In the majority of studies, anoxic or microaerophilic conditions yielded higher activities than fully aerobic conditions
(Table 5.6).In contrast, two studies of seagrass root systems and one of mangrove
sediments found a maximum of root and rhizome-associated nitrogenase activity
under fully oxic conditions. Several studies have reported no observable
difference in activity between samples incubated either oxically or anoxically.
Most recently, we have examined the effect of O2 in muddy and sandy
Table 5.6. Effect of O2 on nitrogenase activity in various marine sedimentary systems
Response
Stimulation by anoxic
or microaerophilic
conditions
Maximum activity under
fully oxic
conditions
No difference between
oxic or anoxic
incubation
System
Sediments
intertidal muds
subtidal muds
carbonate sands
Tha/assia (seagrass)
mangrove
Root/rhizomes
Zostera (seagrass)
Zostera
Spartina (marshgrass)
Spartina
Spartina
Root/rhizomes
Zostera
Ha/odu/e (seagrass)
Sediments
mangroves
Sediments
intertidal
Spartina
Spartina
References
Herbert (1975)
Blake et a/. (1982)
Patriquin and Knowles (1975)
Capone and Taylor (1980a)
Zuberer and Silver (1978)
Capone and Budin (1982)
Brackup and Capone (1985)
Patriquin and McClung (1978)
Patriquin (1978a)
Van Berkum and Sloger (1979)
Smith and Hayasaka (1982b)
Smith and Hayasaka (1982a)
Hicks and Silvester (1985)
Jones (1982)
Whitney et a/. (1975)
Hanson (1977)
102
Nitrogen Cycling in Coastal Marine Environments
Table 5.7. Effect o£Oz on CzHz reduction in muddy carbonate sediments (Corredor and
Capone, 1985)
CzHz reduction
(nmolg-'h-';
1:S.E.)
-Oz
Station
Dl
D2
D3
E2
Date
8 March 1985
13 March 1985
+02
3-7h
5-22 h
3-7h
5-22h
2.39 1: 1.04
2.711:0.135
2.3 1: 0
1.49 1:0.07
1.631: 0.49
1.361:0.01
1.431: 0.07
0.89 1: 0.04
1.541:0.125
1.111:0.12
2.161: 1.54
0.25 1: 0.066
0.781:0.085
0.91 1: 0.08
1.27 1: 0.64
0.32 1: 0.053
carbonate sediments. Quite different effects of Oz on nitrogenase activity were
found in the two sediments (Corredor and Capone, 1985; O'Neil and Capone,
1986). In carbonate muds there was a consistent pattern of Oz sensitivity with
inhibition of up to 83% occurring at some sites (Table 5.7).In contrast, Oz often
had no observable effect in sandy substrate, even for samples collected from
below the oxic-anoxic interface (Corredor and Capone, 1985).
Fine-grained sediments are generally associated with higher organic content,
higher microbial metabolism and lower Oz penetration than are coarser-grained
sediments. It is therefore not surprising to detect N rfixing bacteria adapted to
anoxia in the fine-grained environment and relatively insensitive to Oz in sands.
Muds generally exhibit higher rates of nitrogenase activity than do coarsergrained substrates. Hence, the effect of Oz, along with those factors controlling
Oz penetration into sediments (temperature, rate of consumption, organic load,
bioturbation, etc.) deserve further investigation. Various nitrogenase systems
display different patterns of recovery from Oz inhibition (Postgate, 1982). The
time course and extent of recovery (if any) of sediment Nz fixation after Oz
inhibition has not generally has been considered (Patriquin, 1978a). Investigations along these lines should reveal physiological attributes ofthe diazo trophic
flora of a particular system.
5.5.2 Ammoniumand nitrate
Ammonium control of nitrogenase synthesis has been demonstrated in a
variety of diazotrophic systems (Dixon, 1984; Postgate, 1982), and it often
appears to act in concert with concurrent control of the glutamine synthetase
enzyme system. In broad outline, ammonium may interact with gene products
from the glutamine synthetase gene (gin), which in turn regulate nitrogenase
synthesis. An apparent allosteric control of nitrogenase activity, also through the
glutamine synthetase enzyme system, has also been reported for some photosyn-
Benthic Nitrogen
103
Fixation
thetic (Gotto and Yoch, 1982)and heterotrophic (Ludden et al., 1978)bacteria.
Ammonium additions cause a rapid and reversible 'switch-off' of activity (Zumft
and Castiilio, 1978). A basic enigma of N z fixation in nearshore and coastal
environments is that in fine-grained sediments, which would favor heterotrophic
nitrogenase activity because of lower Oz concentrations and higher organic
content, combined nitrogen levels (ammonium and dissolved organic nitrogen)
are generally quite high and might be expected to repress activity.
Several studies of sediment surface (Van Raalte et aI., 1974;Carpenter et al.,
1978)and sedimentary (Carpenter et al., 1978;Teal et at., 1979)nitrogen fixation
in saltmarsh systems have noted inverse correlations between ambient NH;
concentrations and rates of CzHz reduction. Carpenter et at. (1978) reported a
threshold concentration for inhibition by NH; of 100/lM,while Teal et al. (1979)
concluded that inhibition of sediment N z fixation occurred above 200/lM NH;.
Recently we have seen similar trends in estuarine sediments (Dunham et aI., in
preparation; Figure 5.2) and carbonate muds (Corredor and Capone, 1985).
There was no apparent relationship noted in coarse-grained carbonate sediments
(cf. Patriquin and Knowles, 1975).
Laboratory studies of the effect of ammonium on saltmarsh sediments have
yielded results similar to field-generated correlations ( 200-300 /lM inhibition
threshold) (Table 5.8; Hanson, 1977a; Capone and Carpenter, 1982a).Sediment
ammonium levels are often in excess of these estimated threshold levels of
~
0.3
0.28
0.26
I
'".£:I
0.24
E
u
"0
E
c:
0.2
0.18
0.16
c
0
...
U
:J
"0
0'-
a..
I
0.22
0.14
J
0.12
"'0
0
0
0.1
o
<t
0'''
0
0
0.08
0.06
0.04
"x
~
0
0.02
0
0
Ox 0 0
0
x
x
x
x
x
x
0
x
x
0
0.4
x
jE
'a .ox xXx x
0.8
1.2
1.6
(Thousands)
NH: (fLM)
2
2.4
2.8
Figure 5.2. Relationship between interstitial NH: and nitrogenase activity
in sediments of the Carmens River estuary, New York. All points from seasonal
study of five stations along the salinity gradient. (From Dunham et al., in
preparation.)
104
Nitrogen Cycling in Coastal Marine Environments
inhibition, particularly in fine-grained, organically enriched environments.
Hence one might expect nitrogenase activity to be occurring at submaximallevels
(see below).
An interesting result, further indicating a significant role of ammonium in
controlling rates of Nz fixation in saltmarsh soils, was obtained by Capone and
Carpenter (1982a) and is presented here (Figure 5.3). Sediment cores were
perfused with deoxygenated, acetylene-saturated marsh water which had been
amended with various concentrations of NH:. Ethylene in the effluent stream
was monitored as the index of nitrogenase activity. Rates of activity increased
dramatically when cores were perfused with ammonium-free water, reaching
levels seven or eight times controls with 500 f.1MNH: (about the in situ
concentration of ammonium), and about 70 times the initial, estimated endogenous rate of CzHz reduction. While endogenous nitrogenase activity is measurable
in these systems, it is clearly at levels far below the potential maximum.
We have recently obtained further evidence that observed nitrogenase
activities in sediment systems are below maximum potential rates because of
repression of nitrogenase by NH: (Figures 5.4 and 5.5; Capone, 1984; Capone
and O'Neil, in preparation). L-Methionine-o, L-sulfoximine (MSX), an analog of
'
..
20
'j
>-0
0>
0
E
5
c:
.9.
t;
15
10
"
"0
0
Q:
5
I'"
-
uN
8
.----:::t
16
24
Time (h)
Figure 5.3. EffectofNH: concentration on nitrogenase activity (CzHz reduction) in perfusion
assaysof rhizospheresedimentsof Spartinaalterniflora. Symbols:0 samplesperfusedwithseawater
containing 500Jl.MNH:; . = same containing
400 Jl.MNH:: ., 0 = same at 200 Jl.MNH:; 0 =
same at ambient seawater [NH:] « 5 Jl.M).
Seawater was deoxygenated by gassing with Nz
and 10-15% CzHz. Means of two to four replicate
determinations, :t standard error. (From Capone
and Carpenter, 1982a.)
105
Benthic Nitrogen Fixation
40
35
C>
30
......
0
E
oS
c
.2
....
25
20
0
::J
-0
Q)
0::
t5
N
I
U
to
N
5
0
0
0
20
40
60
80
Time
Control
MSX
0
(h)
NH:
100
"
120
MSX + NH:
Figure 5.4. Effect of L-methionine-D,L-sulfoximine(MSX) on nitrogenase
activity in saltmarsh sediments (11 April 1984).MSX was added to a final
concentration of 10mg/ml; NH: to 10mM
4
3.5
~
C>
......
3
0
E
c
~
0c
.£
-;;;
-0
<:
~
::J ::J
-0 0
CD ~
0:: I-
2.5
2
1.5
N
I
N
U
0.5
0
24
48
139
Time (h)
Figure 5.5. Effect of MSX on nitrogenase activity in sediments of the
temperate seagrass Zostera marina. Sequence of bars: Control, + MSX,
+NH;, +MSX+NH;.
106
Nitrogen Cycling in Coastal Marine Environments
L-glutamateand inhibitor ofglutaminesynthetase,has the recognizedability to
derepress nitrogenase synthesis in the presence of ammonium (Gordon and Brill,
1974; Shanmugam et ai., 1978). MSX caused marked stimulation in observed
nitrogenase activity in saltmarsh and seagrass sediments, even in the presence of
exogenously supplied ammonium (Figures 5.4and 5.5).Yoch and Whiting (1986)
have presented evidence for 'switch-off' control of nitrogenase activity in Spartina
rhizosphere sediments, including the effect of MSX in preventing NH:
'switchoir.
Nitrate does not generally accumulate to significant levels in sediments.
However, several studies have considered the effect of nitrate on nitrogenase
activity in sediments (Table 5.8).In general, concentrations of about 50-200 11M
have an inhibitory effect in a variety of systems. The mechanism for N03
inhibition of nitrogenase is less clear than that for ammonium, and has been the
subject of minor debate. Nitrate can be reduced in the environment to
ammonium through both assimilatory and dissimilatory pathways (Hattori,
1983).Hence, observed inhibition by nitrate may be a result of its conversion to
ammonium. Dicker and Smith (1980b)have suggested an alternative hypothesis,
Table 5.8. Effect of inorganic nitrogen on nitrogenase activity in various marine
sedimentary systems (experimental dosing unless otherwise noted)
Concentration
Response System
Stated
Repressed by NO;
Spartina roots
0.1mM"
Tall and short Spartina
Spartina and sediments
> 25 mg/mI"
20 J1mol/g wt
sediment"
Zostera sediments
Thalassia sediments
100 J1M"
50 J1M
Repressed by NH:
Spartina, surf. sedimentsb
Spartina sedimentsb
Short Spartina
Spartina sediments
Spartina sediments
> 25 J1g/ml"
20J1mol/gwt
mud"
200J1M
2-3mM
?
100J1M"
Spartina roots/rhizomes
Mangrove sediments
Zostera sediments
Carbonate sands
24 J1mol/g wet
sediment
a
Lowest concentration used.
b Threshold
estimates
based
on field observations.
J1M
100
300
40000
200
50
100
200
300
50000
200
2-3
100
480
References
Van Berkum and Sloger
(1979)
Hanson (1977b)
Dicker and Smith (1980b)
Capone (1982)
Capone and Taylor
(1980b)
Carpenter et al. (1978)
Teal et al. (1979)
Hanson (1977)
Dicker and Smith (1980b)
Capone and Carpenter
(1982a)
Yoch and Whiting (1986)
Zuberer and Silver (1978)
Capone (1982)
Patriquin and Knowles
(1975)
Benthic Nitrogen Fixation
107
proposing that a competition exists between (assimilatory) nitrate reduction and
N2 fixation for reducing power and energy. This seems unlikely in NH:richjNOi -poor environments such as most sediments.
5.5.3 Organic substrate
The demand by nitrogen fixation for energy and reducing equivalents is high.
Heterotrophic N2 fixation requires a steady source of oxidizable or fermentable
substrates. The quality and quantity of organic carbon in sediments will depend
on the sources, depositional rates and predominant metabolic mode in the
sediment (Capone and Kiene, 1987).While the organic content of a sediment may
be high, much ofthe material is complex detritallignocelluloses and humates, and
not readily metabolized. It has been suggested that sedimentary nitrogen fixation
could, at times, be limited by organic substrate availability. Several investigations
have considered this possibility by examining the effect of organic substrate
additions on nitrogenase activity (Table 5.9).It is obvious from Table 5.9 that a
wide range of both fermentable and oxidizable organic compounds in a variety of
sedimentary environments produce a stimulatory response in nitrogenase
activity. This stimulation is, at times, quite profound. Some differences have been
noted in the particular substrates which promote nitrogenase activity under oxic
compared to anoxic conditions (e.g.Zuberer and Silver, 1978),possibly reflecting
the responsible agents of activity under the two conditions. Whereas C6 sugars
stimulate activity in Spartina sediments, C4 dicarboxylic acids are more effective
in stimulating activity associated with Spartina roots and rhizomes (Boyle and
Patriquin, 1981).This again suggests distinctions in the nature of the microbial
flora. In a few isolated cases organic additions have caused a depression of
nitrogenase activity (Hanson, 1977b; Patriquin and Knowles, 1972).An interesting recent observation was made by Whiting et al. (1986) who show that rootassociated nitrogenase activity responded rapidly and positively to stimulation of
plant photosynthesis in Spartina. They concluded that root-associated activity
was tightly coupled to organic exudates of the roots.
5.5.4 Inhibitors
Nearshore marine sediments are sites of accumulation of a variety of organic
and inorganic contaminants. Many of these compounds reach levels in the
sediments which may be toxic to the indigenous flora. Several recent investigations have considered the potential or apparent effects of sediment toxification
on N2 fixation. For a variety of heavy metals, inhibitory effectswere noted at high
concentrations ( 1000J1.gmetaljg dry sed.)(Slater and Capone, 1984).Knowles
and Wishart (1977) found that neither oil nor several petroleum fractions tested
had any effect on nitrogen fixation in marine sediment; 1,2,4-trimethylbenzene,
however, caused
completeinhibition.Long-term,but not immediate,decreasesin
nitrogen fixation in subarctic shelf sediments exposed to crude oil have been
~
Table 5.9. Effectof organic substrates on Nz fixationin various sedimentsystems
Substratea
System
Estuarine sediment
Intertidal sediment
Carbonate sediment
Coarse
Callianassa
mounds
Spartina sediment
Spartina sediment
Spartina sediment
Spartina sediment
Spartina sediment
Spartina roots/rhizomes
Zostera sediment
Zostera roots/rhizomes
Zostera roots/rhizomes
Zostera roots/rhizomes
Zostera roots/rhizomes
Thalassia roots/rhizomes
Thalassia roots/rhizomes
Mangrove sediment
Effectb
Sucrose
Glu/Ac
Glu
Glu
EtOH/S04-2
Glu
Mannitol
Mannitol
Succ
Lac
Mannitol
Glu
Succ
Lac
++
++++
+++
++++
++++
(+ +)+ +
O
0
++
++
O
0
disaccharides
+++
Glu
Glu/Mannitol
Glu/Fruct/Sucr
Malate
Fum/Succ
Glu
Glu
Glu
Glu/Sucr
Succ
Glu
Glu
Glu/Mannose
Lactate/ Ac/ ucc
++
0
+++
+++
+
+
:t
+
:t
?
?
:t
:t
:t
:t
:t
:t
:t
:t
:t
+
+
5%
5%
5%
5%
+++
+
+
++
+
++
++++
++++
References
OxygenC
+
+
Marsho et al. (1975)
Marsho et al. (1975)
Herbert (1975)
Jones (1982)
Jones (1982)
Patriquin and Knowles (1972)
Patriquin and Knowles (1975)
O'Neil and Capone (1986; in prep)
O'Neil and Capone (1986; in prep)
O'Neil and Capone (1986; in prep)
O'Neil and Capone (1986; in prep)
O'Neil and Capone (1986; in prep)
O'Neil and Capone (1986; in prep)
O'Neil and Capone (1986; in prep)
Hanson
(1977a)
Hanson (1977b)
Dicker and Smith (1980)
Boyle and Patriquin (1981)
Boyle and Patriquin (1981)
Boyle and Patriquin (1981)
Capone (1982)
Patriquin and Knowles (1972)
Patriquin and Knowles (1972)
Capone and Budin (1982)
Capone and Budin (1982)
Patriquin and Knowles (1972)
Patriquin and Knowles (1972)
Zuberer and Silver (1978)
Zuberer
and Silver (1978)
b Glu = glucose;Fruct = fructose;Sucr= sucrose;Ac= acetate;Succ= succinate;Fum = fumarate.
- = inhibition; a = no significant effect; + = up to 2 x stimulation; + + = > 2-10 x stimulation; + + + = > 10-100 x stimulation; + +
a
..L..L
-
1()(\
v et;nlot;An
-000
...,
c
'"
""
;:;
'"\')
"
s.
""
S.
\')
c
.,
[
Co
.,
S.
'"
...,
t"r1
;:;
'"
".
c
;:;
;;1
'"
Co
Benthic Nitrogen Fixation
109
observed, however (Haine et aI., 1981;Griffiths et aI., 1982).Thomson and Webb
(1984) reported no apparent effect of oil treatment on marsh sediment nitrogen
fixation. Heptachlor, phenanthrene, PCP, DDT and endrin caused immediate
inhibition of acetylene reduction in saltmarsh sediments (Slater and Capone,
1987);the same study demonstrated increasingly severe inhibition by PCP and
lindane after several weeks. In contrast, some organic contaminants stimulated
nitrogenase activity in this study (toxaphene immediately and Aroclor 1254after
3 weeks).
5.6 N2 FIXATION IN BENTHIC MARINE ENVIRONMENTS
The occurrence of N rfixing organisms has been well documented in intertidal
and shallow subtidal marine environments. Recent reviews have comprehensively discussed N 2fixation in benthic environments (Capone, 1983b),associated
with seagrasses (Capone, 1983a),marsh grasses (Patriquin, 1982)and in flooded
soils (Buresh et al., 1980).Table 5.10 summarizes the range of reported values for
in situ N 2 fixation for those systems which have been identified as important sites
of this activity.
Cyanobacterial mats in both temperate and tropical environments exhibit
exceptionally high rates of nitrogenase activity. In fact, reported rates for coral
reef algal mats are amongst the highest areal rates of activity reported for any
natural system (Wiebe et al., 1975).Whereas many of the early studies focused
primarily on mats dominated by heterocystous cyanobacteria, several recent
reports have found considerable light-dependent activity in mats composed of
non-heterocystous filamentous forms (Pearson et al., 1979; Stal et al., 1984;
Bautista and Paerl, 1985). Pearson et al. (1979) and Stal and Krumbein (1981)
have isolated N2-fixing Microcoleus spp. and Oscillatoria spp. from their
respective study sites (Table 5.2).An interesting relationship between the extent
ofN 2fixation in coral reef algal mats and fish grazing was reported by Wilkinson
and Sammarco (1983). In their study, highest rates of nitrogenase activity were
found in heavily grazed areas where algal (cyanobacterial) biomass was least and
there was selection for high growth rates.
The epiphytes of several macro algae and seagrasses are reported to have very
high rates of nitrogenase activity (Table 5.10). In most cases these epiphytes
have been found to be cyanobacteria (Capone, 1977; Capone et al., 1977a,b;
Dromgoole et al., 1978; Goering and Parker, 1972; Penhale and Capone, 1981;
Rosenberg and Paerl, 1981). Head and Carpenter (1975) concluded that an
Azotobacter was the primary agent of N 2 fixation on Codiumfragile, however.
Photosynthetically linked N 2fixation is associated with detrital plant material
in shallow environments (Table 5.10; Capone et al., 1979; Gotto and Taylor,
1976; Hicks and Silvester, 1985). Interestingly, anoxyphotobacteria were implicated as the diazotrophic agents in the study of mangrove leaf detritus by
Gotto and Taylor (1976).
110
Nitrogen Cycling in Coastal Marine Environments
Table 5.10. Summary of rates of C2H2 reduction and estimated N2 fixation
Community/System
Algal mats
coral reef
rocky shore
intertidal marsh
Epiphytes
tropical macroalgae
seagrass
Plant detritus
Root/rhizome bacteria
Sediments
shelf < 200 m
shallow, subtidal
shallow, subtidal seagrass
intertidal
intertidal, saltmarsh
Adapted
C2H2 reduction
(nmolg-lh-1)
-
129-2250
0-11 000
100-1 200
0-1 600
0.0005-0.02
0.02-1.1
0-27
-
N 2 fixation
(mgm -2 d -1)
Number of
studies
6-180
3-7
6-55
7
2
4
1-64
0.02-150
0.6-0.8
0.02-35
7
8
8
3
0.07-0.54
0.4-15.3
5.5-82
1.5-4.8
1.6-142
3
4
7
3
6
from Capone (1983, Tables 8.2-8.6).
Nitrogen fixation has been detected on rinsed roots and rhizomes of several
marine macrophytes induding the marsh grass Spartina alterniflora (Boyle and
Patriquin, 1981), and the temperate and tropical seagrasses Zostera marina
(Capone and Budin, 1982;Smith and Hayasaka, 1982a)and Thalassia testudinum
(Capone and Taylor, 1980a).Heterotrophic N 2-fixingbacteria have been isolated
from Spartina alterniflora (Patriquin, 1978b; McClung and Patriquin, 1980),
Zostera marina (Budin, 1981) and Halodule wrightii (Smith and Hayasaka,
1982b; Schmidt and Hayasaka, 1985) roots.
Root/rhizome-associated nitrogenase activity has generally been found to be
optimal at finite O2 concentrations below full atmospheric (Table 5.6; Patriquin,
1978a;Patriquin and McClung, 1978;Van Berkum and Sloger, 1979;Capone and
Budin, 1982)and stimulated by organic carbon additions (Table 5.9; Patriquin
and Knowles, 1972;Boyle and Patriquin, 1981;Capone and Budin, 1982).Roots
and rhizomes serve as a source of O2 and labile organic carbon (Oremland
and Taylor, 1977; Wetzel and Penhale, 1979; Yoch et ai., 1986) in sediment
environments, which are often highly reducing and contain much refractory
organic material. The root/rhizome interface may thus be a very suitable site for
heterotrophic diazotrophs. Several authors have speculated on the possibility of
symbiotic liaisons between macrophyte and diazotrophs (Patriquin, 1978b;
Capone and Budin, 1982;Capone 1983a).It remains to be determined if specific
associations exist and are generally encountered, and if a bidirectional transfer of
metabolites occurs in presumed associations.
In an attempt to establish the relevancy of root-associated N 2 fixation to
111
Benthic Nitrogen Fixation
Table 5.11. 15Nz incorporation into Zostera marina" tissue
Exposure period
Plant portion
24h
48h
48 h (control)
Leaf tips
Leaf base
Sheath
Rhizomes
Roots
0.022
0.015
0.024
0.509
1.187
0.000
0.285
0.307
1.118
4.385
0.018
0.000
0.000
0.007
0.012
a
All values are in atom % excess. Individual plant roots and rhizomes and
attached bacteria were exposed to 15N z in partitioned incubation vessels held
in a temperature-controlled,
illuminated water bath. Prior to the incubation,
seawater was boiled, allowed to cool and re-equilibrate in a sealed flask.
Either freshly generated
15Nz (87% enriched) or Nz (control) from a
compressed gas cylinder was injected through the stopper of the flask as gas
was taken up during cooling. This seawater was then supplied to the lower
chamber of the incubation flask. Unboiled, filtered seawater was used in the
upper chamber. At the termination of the incubation the plants were rapidly
removed and, in filtered seawater, sectioned and dried to a constant weight at
60°C. Samples were then ground up and prepared for 15N or CHN analysis.
Zostera marina nutrient requirements, I examined the extent of transfer of
recently fixed nitrogen in the root zone through the plant (Table 5.11).Relatively
high levels of enrichment in 15N were noted in distal portions of the plant after
exposure of the roots to 15N2' indicating rapid transfer of recently fixed nitrogen.
This provides direct evidence that bacterial N 2fixation can subsidize plant needs.
Shallow sediments have generally been found to exhibit measurable and, at
times, substantial rates of nitrogenase activity (Table 5.10).Vegetated sediments
in subtidal and intertidal areas have particularly high rates of activity as a result
of the presence of the macrophyte and, hence, a source of labile carbon. As
discussed earlier (Section 5.4), organic-rich fine-grained sediments generally
appear to exhibit higher specific and areal rates than do coarse-grained organicpoor sediments, despite the generally higher ammonium concentrations found in
the former. Many of the early studies focused on clastic sediments of temperate
estuarine and coastal areas (summarized in Capone, 1983b). Recently, several
investigations have examined tropical carbonate sediment environments
(Table 5.12). Wilkinson et al. (1984) found that nitrogenase activity in coralline
substrata increased in reefs progressively farther offshore and in more oligotrophic waters. Corredor and Capone (1985)recently examined a number of sites
along the south coast of Puerto Rico and found that high rates of nitrogenase
activity were associated with muddy sediments while sands exhibited activities
which were considerably lower.
While enrichment and enumeration studies have provided an indication of the
range of bacterial physiologies in the sediments (Table 5.1),it is difficult to assess
112
Nitrogen Cycling in Coastal Marine Environments
Table 5.12. Nitrogen fixation in tropical carbonate sediment environments
Location
Substrate
Barbados
sediments
Hawaii
sediments
Puerto Rico
muds
sands
Bermuda
Inshore
fine grained
coarse grained
Offshore
fine grained
coarse grained
Great Barrier Reef
Coralline surface rubble
inner shelf reef
mid shelf reef
outer shelf reef
References
N 2 fixation
(mg N m -
2
d-
1)
Patriquin and Knowles (1972)
0.9
Hanson and Gunderson (1977a)
1.7
Corredor and Capone (1985)
2.2-4.3
0.13-1.21
O'Neil and Capone (1986; in prep)
3.4
0.74
0.86
0.31
Wilkinson et al. (1984)
0-0.56
0-1.53
0-4.28
Table 5.13. Estimates of nitrogen input by N2 fixation in various systems
System
N2 fixation
mgNm-2d-1
Coral reef (Smith, 1984)
0.9-1.6
Shark Bay
2.4-8.2
Christmas Island
6.7-26
Canton Atoll
Baltic Sea
(Gundersen, 1981)
Narragansett Bay
(Nixon and Pilson, 1983)
Charleston Pond
(Nixon and Pilson, 1983)
Saltmarshes
Sippewissett Marsh (Valiela, 1983)
Gulf Coast
(DeLaune and Patrick, 1980)
Thalassia (seagrass)
82
Patriquin and Knowles (1972)
21
Capone and Taylor (1980a)
Zostera (seagrass)
5.5
(Capone, 1982)
gNm-2y-l
0.3-0.6
0.9-3.0
2.4-9.5
Percentage of
plant N-demand
52-184
37-127
31-121
0.6
3
0.01
0.02
0.42
0.76
6.8
82
15.4
73
> 100
20-50
3-28
113
Benthic Nitrogen Fixation
the importance of bacterial isolates to observed in situ activities. Using specific
inhibitors, Capone et at. (1977b), Nedwell and Aziz (1980), Capone and Taylor
(1980b) and Capone (1982), working in seagrass and saltmarsh sediments,
concluded that sulfate-respiring bacteria were responsible for the bulk of the
sedimentary nitrogenase activity in reducing environments. This observation
deserves further study.
Estimated contributions of nitrogen fixation for a variety of defined systems
are presented in Table 5.13. The studies of saltmarsh and seagrass communities
have all directly examined nitrogenase activity in situ and have related N z
fixation to estimated or empirically determined macrophyte demands. In all cases
a large fraction of the plant requirement appears to be satisfied by bacteri~l Nz
fixation. In contrast, nitrogen fixation appears to playa relatively minor role in
the nutrient-rich Narragansett Bay, in a coastal pond in Rhode Island and in the
Baltic Sea. Direct observations in Narragansett Bay and the Baltic were relatively
limited, while the estimate for Charleston Pond is calculated from literature
values. Smith (1984) has estimated upper and lower values for Nz fixation in
several tropical environments with restricted and quantifiable nutrient inputs
using a model relating empirically determined inputs and distributions of
nitrogen and phosphorus, and plant and sediment N:P ratios. At all three sites
considered, nitrogen fixation accounted for a large portion ofthe estimated plant
uptake. Areal rates of nitrogen fixation calculated from the model agree well with
observed activities in carbonate environments (Table 5.12),more so if cyanobacterial mat N z fixation (common in such environments) is considered (Table 5.10).
A number of authors have attempted to estimate the annual integrated
contribution ofNz fixation to the oceanic nitrogen cycle (Table 5.14).While the
data base for these estimates has increased substantially with time, there is
Table 5.14. Estimates of the total annual contribution of combined nitrogen by marine
N z fixation
Total oceanica
References
Burns and Hardy (1975)
Soderlund and Svensson (1976)
Paul (1978)
Fogg (1982)
Capone and Carpenter (1982b)
a Tg=IO'2g
Subsystem
(Tg Njy)
Sediments
Saltmarsh
10-60
2
0-2oom sediment
Bare estuary
Seagrass
Coral reefs
Saltmarsh
Mangroves
Total benthic
2.7
0.43
1.5
2.8
6.3
1.5
15
35
30-130
36
15
20
114
Nitrogen Cycling in Coastal Marine Environments
surprisingly little variance in the range of estimates (save for the upper limit by
OneconclusionofCaponeand Carpenter(1982b)
is
Soderlund and Svenson, 1976).
that the majority of nitrogen input occurs in shallow benthic environments over a
relatively small area of the sea as a whole. The suggestion by Martinez et al. (1983)
of a potential underestimate of pelagic Nz fixation because of sample manipulation deserves further investigation. Small, previously overlooked inputs of
nitrogen over the expanses of the sea could easily exceed benthic inputs.
Nitrogen fixation in the sea as presently estimated appears to be comparable to
nitrogen input through river flow (~13 to 35 Tgjy; Walsh et aI., 1981), and
considerably less than oceanic denitrification (74 to 94 Tgjy; Hattori, 1983).
Integrated annual N z fixation accounts for about 0.3% of total phytoplankton
uptake. Since the source of most of our in situ estimates of N z fixation,
particularly in sediments, derives from CzHz reduction determinations the
foregoing estimates must be viewed with caution. It is encouraging that modeled
estimates ofNz fixation required to balance nitrogen budgets in three restricted
ecosystems (Smith, 1984) appear to agree well with extrapolations from CzHz
reduction-based field measurements (Tables 5.10 and 5.12). However, further
examination of the appropriateness of the CzHz reduction method, and
determination of the relationship between CzHz reduction and actual N z fixation
in a variety of benthic systems, is required.
5.7 CONCLUSIONS
Diazotrophic bacteria are easily obtained and nitrogen fixation detected in a
wide range of shallow benthic marine environments. The CzHz reduction
method has been used most commonly to assess nitrogenase activity in situ.
While it certainly appears to be a good qualitative and relativistic measure of
nitrogenase activity in marine sediments, there is concern over its quantitative
accuracy because of the paucity of comparisons with direct methods of assessing
Nz fixation. In general, workers have assumed ratios ofCzHz reduced to Nz fixed
gleaned from other systems (i.e. 3:1 to 4:1), but more direct comparisons
with appropriate direct methods are required in order to validate present
extrap91ations.
For heterotrophic Nz fixation in the sediments, oxygen concentration,
ammonium concentration and organic substrate availability can all, to varying
extents, affect the rate of nitrogen fixation. Among shallow benthic systems
nitrogen fixation appears to be particularly important in saltmarsh, seagrass and
coral reef ecosystems. However, nitrogen fixation is only a minor component of
the nitrogen cycle of unvegetated sediments, although some activity is detectable.
Unless major new sources of planktonic N z fixation are uncovered, it is
concluded that shallow benthic areas are the main sites of nitrogen fixation in the
ocean.
115
Benthic Nitrogen Fixation
ACKNOWLEDGEMENTS
I thank Ron Oremland, Jim Bauer and Jennifer Slater for their reviews and
comments on the text, and Jennifer for her excellent editorial assistance and for
help on the MSX experiments. The National Science Foundation (Grants OCE82-00157. OCE-84-17595, OCE-85-15886) provided support for travel to the
symposium as well as for much of the research. Research support from the US
Environmental Protection Agency (Grant R-8094-75-01-0), the New York Sea
Grant Institute and the Puerto Rico Sea Grant Office of The National Oceanic
and Atmospheric Administration and the Hudson River Foundation (Grant 1483B-12) is also gratefully acknowledged.
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