Freshwater Biology (2004) 49, 690–708
Do cyanobacteria dominate in eutrophic lakes because
they fix atmospheric nitrogen?
L. R. FERBER,* S. N. LEVINE,* A. LINI† AND G. P. LIVINGSTON*
*Rubenstein School of Environment and Natural Resources, University of Vermont, Burlington, VT, U.S.A.
†
Department of Geology, University of Vermont, Burlington, VT, U.S.A.
SU M M A R Y
1. The sources of nitrogen for phytoplankton were determined for a bloom-prone lake as a
means of assessing the hypothesis that cyanobacteria dominate in eutrophic lakes because
of their ability to fix nitrogen when the nitrogen : phosphorous (N : P) supply ratio is low
and nitrogen a limiting resource.
2. Nitrogen fixation rates, estimated through acetylene reduction with 15N calibration,
were compared with 15N-tracer estimates of ammonium and nitrate uptake monthly
during the ice-free season of 1999. In addition, the natural N stable isotope composition of
phytoplankton, nitrate and ammonium were measured biweekly and the contribution of
N2 to the phytoplankton signature estimated with a mixing model.
3. Although cyanobacteria made up 81–98% of phytoplankton biomass during summer
and autumn, both assays suggested minimal N acquisition through fixation (<9% for the
in-situ incubations; <2% for stable isotope analysis). Phytoplankton acquired N primarily
as ammonium (82–98%), and secondarily as nitrate (15–18% in spring and autumn, but
<5% in summer). Heterocyst densities of <3 per 100 fixer cells confirmed low reliance on
fixation.
4. The lake showed symptoms of both light and nitrogen limitation. Cyanobacteria may
have dominated by monopolizing benthic sources of ammonium, or by forming surface
scums that shaded other algae.
Keywords: ammonium uptake, cyanobacteria, N : P ratio, nitrate uptake, nitrogen fixation, stable
isotope
Introduction
Cyanobacterial blooms are a frequent and unwelcome
consequence of lake eutrophication. With rising nutrient inputs, the relative abundance of cyanobacteria
increases, while first chrysophytes and cryptophytes,
and then chlorophytes and diatoms, diminish in
importance (Watson, McCauley & Downing, 1997).
The cyanobacterial species involved are relatively
large-celled, filamentous or colonial, and contain gas
vacuoles for buoyancy regulation. Many, but not all,
have heterocysts for N2 fixation in oxic well-lit waters.
Correspondence: S. N. Levine, Rubenstein School of
Environment and Natural Resources, University of Vermont,
Burlington, VT 05405, U.S.A. E-mail: [email protected]
690
The surface scums that these algae form during calm
weather are unsightly, odiferous, and sometimes toxic
(Reynolds, 1984). Anxious to prevent or reduce
blooms, lake managers have sought understanding
of the conditions that lead to their dominance. More
than a dozen hypotheses have been put forth [see
reviews of Shapiro (1990) and Hyenstrand, Blomqvist
& Pettersson (1998a)], the more convincing of which
suggest that the nature of resource limitation changes
during the eutrophication process, promoting cyanobacteria from a poorly- to a highly-competitive position. Phosphorus limitation generally is accepted as
the initial condition, while the hypotheses differ in
identifying nitrogen, CO2 or light as the limiting
resource under eutrophy. In addition, some hypotheses suggest that, once all resource limitations are
2004 Blackwell Publishing Ltd
Cyanobacterial dominance and nitrogen fixation 691
relieved, algae grow at their intrinsic rate of increase
and relative abundance is determined by differential
mortality. While the bloom-forming cyanobacteria
grow slowly, their grazing mortality is minimal
because of large size, low lipid content and frequent
toxicity (Lampert, 1987), and sinking losses are substantially reduced by vacuolation (Knoechel & Kalff,
1975).
Here we provide a test of the most widely accepted
hypothesis about cyanobacterial dominance in eutrophic lakes, the low nitrogen : phosphorous (N : P)
hypothesis. First proposed by Pearsall (1932), and later
elaborated on and popularised by Schindler (1977) and
Smith (1983), this hypothesis maintains that cyanobacteria are masters of survival in environments poor in N
and that their dominance in eutrophic systems is
related to the low N : P ratios of major anthropogenic
pollutants (e.g. animal and human wastes). The unusually high demand to supply ratios of P and N suggest
that these nutrients are the basis of most phytoplankton
competitions. Pearsall (1932) noted an association
between cyanobacterial presence and low N : P ratios
in English lakes but knew too little about algal physiology to provide a convincing explanation. He speculated that, as prokaryotes, cyanobacteria may use more
organic N than other algae. Schindler (1977) proposed
the mechanism that is now an integral part of the
hypothesis, that cyanobacteria gain dominance by
fixing atmospheric nitrogen when water column supplies of dissolved inorganic N (DIN) are minimal and
populations of eukaryotes (which cannot fix) severely
N limited. Because the atmospheric supply of nitrogen
is vast, substrate limitation of nitrogen fixation is not
expected. Cyanobacteria exploit the rising P inputs no
longer available to eukaryotes and match P uptake with
nitrogen fixation, biomass ultimately being limited by
self-shading. Schindler (1977) based this mechanism on
observations made at the Experimental Lakes Area
(ELA) in Ontario, Canada. Lakes experimentally
enriched with N and P at ratios above the demands of
phytoplankton developed chlorophyte dominants,
while blooms of heterocystous Anabaena and Aphanizomenon occurred when the enrichment ratio was
lower. Smith (1983) further promoted the hypothesis
by showing that the relative abundance of cyanobacteria in 17 north temperate lakes correlated negatively
with total N : total P (TN : TP) ratio.
Previous tests of the low N : P hypothesis continued the field enrichment studies and empirical ana 2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
lyses initiated by Schindler (1977) and Smith (1983),
but with less straightforward results. Nitrogen additions to Manitoban and British Columbian lakes with
blooms of Aphanizomenon and Anabaena, respectively,
successfully reduced the biomass of these fixers, but
the new dominants were Microcystis and Synechoccus,
non-heterocystous (non-nitrogen-fixing) cyanobacteria, rather than the chlorophytes favoured in ELA
lakes (Barica, Kling & Gibson, 1980; Stockner &
Shortweed, 1988). Microcystis is a particularly toxic
bloom-former, and thus was no improvement over
Aphanizomenon. Schindler (1977) overlooked non-fixing cyanobacteria in his initial formulation of the low
N : P hypothesis because they were not major
respondents to whole-lake nutrient enrichment at
the ELA. However, Levine & Schindler (1999) later
urged the scientific community to restrict the low
N : P hypothesis to prediction of dominance by
heterocystous species, having shown in mesocosm
experiments that several non-heterocystous cyanobacterial taxa (Oscillatoria, Pseudoanabaena and Lyngbya)
increased in relative abundance along an N : P gradient. Unfortunately, most empirical studies have dealt
with total cyanobacterial biomass. The result has been
reports of positive (e.g. McQueen & Lean, 1987) as
well as negative (Smith, 1985; Smith, Willén &
Karlsson, 1987) relationships when data sets have
been relatively small, and a finding of no relationship
when more than 100 lakes have been included (Harris,
1986; Canfield, Philips & Duarte, 1989).
The more recently formulated low DIN hypothesis
of Blomqvist, Pettersson & Hyenstrand (1994) and
Hyenstrand et al. (1998a,b) suggests that heterocystous versus nonheterocystous cyanobacterial dominance is determined by whether there is a benthic
ammonium source available during water column
DIN depletion. Vacuolated filamentous or colonial
cyanobacteria such as Microcystis and Oscillatoria can
migrate vertically with little energy expenditure and
with greater speed than flagellated algae (Reynolds,
1984). Bringing stored N back to the surface, they may
grow more quickly than heterocystous species that
have stayed in the epilimnion and fixed nitrogen.
Dominance by fixers is predicted only when both
epilimnetic and benthic DIN sources are inadequate to
meet demands. A shortcoming of the low DIN
hypothesis is its failure to acknowledge that most
heterocystous cyanobacteria also are vacuolated and
colonial. They may be as likely to migrate as non-
692
L.R. Ferber et al.
heterocystous forms, and thus may dominate both
when there is a benthic N source and when fixation is
required to obtain adequate N.
Our test of the low N : P hypothesis was unique,
although simple. Inverse logic dictates that N2 must
be the principal species of N used by heterocystous
cyanobacteria during blooms if these algae have
gained and maintained dominance through fixation.
Consequently, we assessed N sources in a hypertrophic lake over the course of an ice-free season,
looking for a relationship between periods of heterocystous cyanobacterial dominance and dependency
on atmospheric nitrogen. Three independent assays
contributed to our conclusions, monthly assessments
of ammonium, nitrate and N2 incorporation rates
through in-situ incubations with 15N tracers and
acetylene (referred to as the ISI assay), biweekly
natural N stable isotope analysis (NSID assay), and
biweekly determination of heterocyst density on fixer
filaments. The last variable was useful because
cyanobacteria with access to ammonium produce
few heterocysts, ensuring that the high energetic cost
of fixation (12 moles of ATP per mole of nitrogen
fixed) is not incurred unnecessarily (Paerl et al., 1981).
Consequently heterocyst density is a good indicator of
reliance on atmospheric nitrogen. To allow for evaluation of hypotheses about cyanobacterial dominance
other than the low N : P hypothesis, we measured
light availability throughout the study and examined
biweekly water chemistry (N, P and inorganic C
forms) and zooplankton populations.
concentration, 88 lg L)1 ; and Secchi depth, 0.78 m
(N. Kamman, Vermont Department of Environmental
Conservation, pers. comm.). The mean TN concentration for 1997–99 was 124 lM (Lescaze, 1999; this
study).
Macrophyte beds (primarily Potamogeton crispus L.)
cover much of the lake bottom in spring and early
summer (DeYoe, 1981; our observations). The winter
and spring phytoplankton communities are dominated by diatoms and chlorophytes (DeYoe, 1981; our
observations). Vacuolated cyanobacteria appear in
May, and generally by June reach bloom densities.
Aphanizomenon flos-aquae L. Rolfs, a nitrogen fixer, is
the most frequent dominant, but this role is sometimes
assumed by Anabaena species (including A. flos-aquae
(Lyngby) Brébisson, A. circinalis Rabenhorst, and A.
planctonica Brunnth.), or by non-heterocystous Planktothrix agardhii (Komarek) Anagnostides (probably
what DeYoe (1981) called Oscillatoria subbrevis Schmidle) or M. aeruginosa Kuetz. emend. Elenkin (DeYoe,
1981, Lescaze 1999, current study). The zooplankton
community consists of a mixture of cladocerans
(especially Daphnia galeata mendotae Birge and Chydorus sphaerida O.F. Müller), copepods (mostly Acanthocyclops vernalis Fischer and Diaptomus minutus
Liljeborg) and rotifers (e.g. Brachionus, Keratella, and
Filinia) (Schuyler, 1972; this study). Yellow perch
(Perca flavescens Mitchell) and golden shiner (Notemigonus crysoleucas Mitchell) are the most abundant fish
(B. Chipman, Vermont Fish and Wildlife, pers. comm).
Nitrogen fixation
Methods
Study site
Shelburne Pond, 16 km southeast of Burlington, VT,
U.S.A. (4423¢N, 739.5¢W), was the study site. Shallow
(mean depth 3.4 m; maximum depth 7.6 m) and
moderately sized (176 ha), this lake is ice-covered in
winter (December to April) and polymictic in summer. An epilimnion approximately 1 m in depth
develops between May and October, but is destroyed
periodically (typically 3–4 times per year; DeYoe,
1981) by storm mixing. Agricultural runoff and
soluble P-rich dolomite in the 20-km2 catchment
create hypertrophic conditions. For the period
1977–98, spring and summer TP concentrations
averaged 3.3 and 5 lM , respectively; chlorophyll a
Nitrogen fixation in the lake was estimated monthly
using the methods and numerical model of Levine &
Lewis (1987). Integrated epilimnetic water samples
were incubated with acetylene in 50-mL glass syringes
suspended from a floating frame at depths of 0.02, 0.5,
1.0, 1.5 and 2.0 m for 3 h (all analyses done in
duplicate). The ethylene produced was measured on
a Shimadzu GC14A gas chromatograph (Shimadzu,
Kyoto, Japan) with a Hayesep T pre-column and S
primary column, and a flame ionization detector.
Temperatures for the column, injector and detector
were 45, 100 and 120 C, respectively. ‘Calibration’
factors for conversion of the acetylene reduction (AR)
values to nitrogen fixation rates were obtained by
incubating duplicate surface samples (0.02 m) with
15
N2, determining the isotopic content of phytoplankton
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
Cyanobacterial dominance and nitrogen fixation 693
collected on pre-combusted GF/C glass fibre filters,
and comparing the results with AR values for the
same depth.
Nitrogen fixation is known to be a highly lightsensitive process, as light provides both reductant and
ATP for the splitting of the triply bound N atoms (Paerl
et al., 1981). Light inputs during incubations were
estimated for each of the five incubation depths from
incident solar radiation (I0) measured on shore with a
LiCor Li-190 SA light meter (LI-COR, Lincoln, NB,
U.S.A.), and the light extinction rate, using the equation:
Iz ¼ I0 eez ;
ð1Þ
where Iz is light at depth z; I0 is light at the surface;
and e is the light extinction coefficient measured with
a LiCor Li-192SA submersible probe. The relationship
between fixation rate and light availability was determined using the equation:
Ni ¼ Ns Ns ea þ D;
ð2Þ
where a ¼ aINs1 . Ni is the fixation at light intensity I
(Ei m)2 h)1); Ns is fixation at light saturation; D is
fixation in the dark; and a is the slope of the rising limb
of the relationship between fixation and light intensity.
N, Ns and D may have the units nanomol N L)1 h)1
(volumetric fixation rate) or nanomol N (million
heterocysts))1 h)1 (heterocyst-specific fixation rate).
The numerical model first determined average light
intensities for 0.1-m depth intervals within the lake
hourly, and then used the light response relationship
to determine volumetric rates. These were multiplied
by layer volumes and integrated over the depth of the
epilimnion (1 m) and the 5-day period to arrive at
molar estimates for the whole epilimnion.
Ammonium and nitrate uptake
Nitrate and NHþ
4 uptake rates generally were measured within a day of the N fixation assay. 15N-labelled
tracer (approximately 98 atom per cent enrichment,
Cambridge Isotope Laboratories, Andover, MA,
U.S.A.) was added to 300 mL glass bottles as NH4Cl
(0.83 lM ) or NaNO3 (0.13 lM ) and the bottles incubated in the lake at a depth of approximately 33 cm. The
incubated bottles were filtered in the field over a time
course (generally after 0, 0.5, 1, 2, 4 h) onto precombusted GF/C glass fibre filters. The filters were ovendried at 60 C, sealed in quartz tubes containing CuO
(3.0 g), CaO (approximately 0.5 g), and Cu (2.5 g) and
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
combusted at 900 C (Kendall & Grim, 1990). The
resulting nitrogen gas was analysed to determine its
15
N/14N ratio on a VG/Isogas Sira II isotope ratio
mass spectrometer (VG/Isogas, Middlewich, U.K.).
The following equation from Fisher et al. (1988) was
used to calculate the rates of uptake:
U¼
dðatom%P15 NÞ
dt
atom %
PN
15 NHþ
4
or
15 NO
3
;
ð3Þ
)1
)1
where U ¼ uptake rate in lmol L h , d(atom %
P15N) is the change of the isotopic composition of the
PN pool over time); PN is the size of the particulate
15
nitrogen pool; and atom per cent 15NHþ
4 or NO3 is the
isotopic composition of the N pool at the midpoint of
the experimental time interval. Only the initial linear
portion of the labeling curve (before isotope dilution
through regeneration reduced the curve slope) was
used to calculate d(atom per cent P15N)/dt.
A problem with using stable isotopes as tracers is that
their detection requires fairly substantial isotope additions, sufficient to augment uptake rate if the process is
substrate limited. We sought to minimize this effect by
keeping our additions at approximately 10% of what
had been mean ambient values in past years. However,
DIN concentrations in summer 1999 were lower than
expected, and tracer additions raised substrate concentrations by as much as 100%. Consequently, the
estimates reported here [what we will call direct tracer
(DT) estimates] must be viewed as potential, rather
than ambient, rates. Their usefulness is mainly in
assessing whether uptake was substrate limited. Nitrogen-sufficient algae should take up N near the maximum uptake velocity (Vmax) with and without
substrate addition, as in both cases uptake rate is
determined by enzyme activity or other physiological
variables, not substrate concentration (Levine, 1989).
Rates of substrate uptake at ambient concentrations
may be obtained by determining Michaelis–Menten
relationships for the processes and solving the equations for ambient substrate concentrations (Dowd &
Riggs, 1965). We carried out this procedure on 19
August. Water samples were treated with 15N-label
and either KNO3 or NH4Cl at six different concentrations (0, 0.07, 0.15, 0.36, 0.71 and 1.79 lM ) and
incubated at a depth of 0.33 m for 4–5 h. Rate
constants for uptake were obtained from the difference in particulate 15N content at the beginning and
end of the incubation period. To facilitate the use of
694
L.R. Ferber et al.
the relationship to estimate ambient uptake on dates
other than 19 August, we normalised the equation to
chlorophyll a. Consequently, the following equation
was used to acquire what we refer to as Michaelis–
Menten (MM) estimates of ambient uptake rates:
c
Uac ¼ Umax
S
chl:;
Km þ S
ð4Þ
where Uac is the ambient uptake rate (lmol L)1 h)1);
c
Umax
is the maximum uptake velocity [lmol (lg
chlorophyll a L)1) h)1); S is the substrate concentration
(lM ); Km is the half-saturation constant (lM ); and chl. is
the chlorophyll a concentration (lg L)1). Because
temperature changed little over the summer and
cyanobacteria were consistently dominant, we believe
the equation obtained on 19 August was roughly
accurate for the summer period. In spring and late
autumn, however, temperatures were lower and diatoms dominant, so the equation was inappropriate.
Fortunately, substrate concentrations were high during these periods, so that the direct tracer measures are
probably good estimators of ambient rates.
On 20 August, we examined the relationship
þ
between NO
3 and NH4 uptake rates and light
intensity. Samples were treated with 15N-labelled
substrate and incubated at four depths (0.02, 0.25,
0.5, 1 or 2 m) in the lake for 4–5 h. The light intensities
experienced at each depth were determined using
solar radiation and light extinction data obtained with
a LiCor light meter, and use of eqn 1. The generation
of light response curves for ammonium and nitrate
uptake was not necessary, however, as light-stimulated uptake was not observed (see below). Thus, whole
epilimnion uptake rates were obtained by simply
multiplying the volume of this layer by the volumetric
uptake rates measured at 0.33 m.
Natural stable isotope distribution
Determination of the relative contributions of DIN
and nitrogen fixation to phytoplankton N nutrition
from the distribution of N isotopes in lakes is possible
because the isotopic signatures of NHþ
4 and NO3
generally differ from that of atmospheric nitrogen.
The phytoplankton signature reflects the ‘mix’ of
source use. Specifically,
PC ¼
ðd15 Ni fi Þ d15 Np
ðd15 Ni fi Þ ðd15 Na fa Þ
100;
ð5Þ
where PC is the percent contribution of nitrogen
fixation to N incorporation; d15Ni, d15Np, and d15Na
are the isotopic signatures of DIN, phytoplankton and
atmospheric N2, respectively; and fi and fa correct for
isotopic fractionation during DIN uptake and nitrogen fixation, respectively (modified from Shearer &
Kohl, 1989). Delta 15N is defined as
d15 Nð&Þ ¼
ð15 N=14 NÞsample ð15 N=14 NÞstandard
ð15 N=14 NÞstandard
ð6Þ
1000;
where the standard is atmospheric nitrogen (the
signature of which is defined as 0&). d15Ni is
generally a composite variable, as NHþ
4 and NO3
may have different isotopic signatures. However, we
þ
use only the value of d15NHþ
4 here because NH4
concentrations typically were much greater than NO
3
concentrations, and uptake rates for ammonium were
much greater than those for nitrate (see below). We
assumed that fa was )2&, as this is the amount of
fractionation normally reported for cyanobacteria
growing in culture with N2 as their sole N source
(Wada & Hattori, 1976; Macko, Estep & Hoering, 1982;
Gu & Alexander, 1993b; Lescaze, 1999). The value of fi
is dependent on substrate concentration when algae
are not N limited, but approaches 0& with substrate
limitation (Fogel & Cifuentes, 1993; Lescaze, 1999).
Fogel & Cifuentes (1993) found 0& fractionation
during ammonium uptake by marine diatoms at
concentrations <20 lM . We used a more conservative
threshold for the decision to assume no fractionation,
ammonium concentrations <5 lM , plus other symptoms of N limitation. We chose not to use the model in
spring and autumn, when concentrations exceeded
the threshold because relationships between fi and
substrate concentration have not been determined for
lacustrine species.
We collected water for natural stable isotope distribution analysis from the epilimnion of Shelburne
Pond biweekly during the ice-free season of 1999
(May to November), and then monthly from December to February. Samples were taken on or within
2 days of the ISI assessments. Integrated duplicate
samples were obtained by lowering a 5-cm diameter,
1.83-m long PVC pipe into the water, stoppering it,
and allowing the water to drain through a 150 lm
sieve (to remove macrozooplankton) into 20-L plastic
containers. Within 3 h of sample collection, seston
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
Cyanobacterial dominance and nitrogen fixation 695
was separated from the water through filtration onto
pre-combusted Whatman GF/C glass fibre filters
(nominal pore size 1.2 lm). These filters were ovendried (60 C) and analysed for 15N/14N ratios using
the method of Kendall & Grim (1990) described
above. The isotopic signature obtained was assumed
to be that of phytoplankton, although the seston
retained on filters inevitably includes detritus and
some microzooplankton and heterotrophic bacteria.
The water used in d15N analysis was filtered
through Gelman Aquaprep 600 groundwater filters
(pore size 0.45 lm) and stored frozen. Isotope extraction on thawed samples was according to the procedure of Downs et al. (1999). Ammonium and nitrate
(2–15 lmol) were collected onto DOWEX-50W and
DOWEX-1 resins, respectively, by pumping or gravity-dripping water through 2 cm diameter columns at
a flow rate of 1.5 mL min)1. Samples were ‘dripped’
for an average of about one day, and samples with so
little N present that more than 4 days were required
to process them were discarded because of concerns
about microbial contamination and other factors. The
concentrated N was eluted with 2 M KCl, and then
gathered onto precombusted and acidified 5 mm
diameter GF/C filters by placing the solution and
packets containing filters in sealed Nalgene bottles
along with MgO (2 g L)1) and, in the case of nitrate
extraction, Devarda’s Alloy (4 g L)1), and agitating
the solution on a shaker table (speed 100 rpm) for
7 days. The filters were dried in the presence of
concentrated sulphuric acid (approximately 60 C),
and sealed in tin capsules (5 by 9 mm) prior to
combustion and analysis for 15N abundance on a
Finnigan Delta isotope ratio mass spectrometer (run
by R. Michener at Boston University). Although
Downs et al. (1999) reported minimal isotopic fractionation during the extraction of ammonium from
water, we included NH4Cl standards in our analyses
to detect and quantify fractionation rates.
Phytoplankton and heterocysts
Samples for phytoplankton and heterocyst counts
were preserved in the field with acid Lugol’s
solution. Phytoplankton was concentrated in settling
chambers and measured and counted under an
Olympus CK2 inverted microscope (Olympus, Tokyo, Japan) at 400· magnification (Utermöhl, 1958).
At least 100 fields or 100 heterocysts were counted
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
for heterocyst density determination, but only five
fields were counted for determination of species
composition (these counts included >100 cells).
Zooplankton samples were collected with a 64-lm
mesh net hauled through the depth of the water
column, and preserved with sugar-buffered formalin
(Haney & Hall, 1975). Species were identified and
counted under the inverted microscope.
Other measurements
To determine whether the uptake results obtained
were realistic, primary production rates were measured on 20 August and multiplied by the C/N ratio
of seston (4.98 by weight) to arrive at estimates of N
demand. The method of Fee et al. (1992) was used to
estimate whole-lake primary production from a light
response curve, data on light availability in the lake
and bathymetry. The light response curve was
obtained by incubating duplicate light and dark
bottles at four depths (0.3, 0.6, 1.0 and 1.4 m) for 4 h
and estimating CO2 fixation from the change in
dissolved oxygen concentrations (Wetzel & Likens,
1991). Light data were obtained as for nitrogen
fixation. Particulate C and N were determined
through combustion of seston collected on precombusted GF/C filters in a CE Instruments C/N
analyser. These results did not take into account
phytoplankton respiratory losses at night. Consequently, we used the increase in phytoplankton
biomass between our samplings on 10 and 19 August
to provide a second estimate of phytoplankton N
demand. Phytoplankton was assumed to have a dry
weight of 0.47 pg lm)3, a C content of 50%, and a
C : N ratio of 6 : 1 by weight (Reynolds, 1984).
Temperature, pH, alkalinity, Secchi depth, light
extinction rate, chlorophyll a concentration, and the
concentrations of total nitrogen (TN), ammonium-N,
nitrate-N, total phosphorus (TP) and soluble reactive
phosphorus (SRP) were measured during each of the
natural stable isotope distribution samplings. Water
was collected with the integrated sampler and filtered
through GF/F glass fibre filters to separate particulate
and dissolved phases. Chlorophyll was extracted in hot
ethanol, and its colour intensity measured on a Shimadzu 1600 UV/VIS spectrophotometer (Sartory &
Grobbelaar, 1984). Lorenzen’s method of acid addition
(Lorenzen, 1967) was used to correct for phaeophytin
pigments. Ammonium, nitrate/nitrite and phosphate
696
L.R. Ferber et al.
were analysed on a Lachat flow injection autoanalyser
(QuickChem 8000) using methods described by Patton
& Crouch (1977); Anderson (1979) and Murphy & Riley
(1962), respectively. Alkalinity was determined by
sulphuric acid titration with phenolphthalein and
bromcresol green-methyl red indicators (Rainwater &
Thatcher, 1960), and used along with pH to calculate
dissolved inorganic carbon (DIC) and free CO2 concentrations (Wetzel & Likens, 1991).
Statistical analyses
Use of a mixing model to determine the relative
contributions of atmospheric nitrogen and DIN to
phytoplankton nutrition requires that the two sources
have distinct isotopic signatures. We used a t-test
(P ¼ 0.05) to confirm that the mean d15N value of
NHþ
4 was significantly different from )2&, the normal value for phytoplankton totally reliant on fixation
as a N source. One-way A N O V A using the Student–
Newman–Keuls (SNK) test was used to test for
differences in NHþ
4 uptake rates during the lightuptake experiment. To investigate the role of envi
ronmental parameters on NHþ
4 and NO3 uptake and
N2 fixation rates, and on natural stable isotope
distribution results, Pearson product moment correlation analysis (Darlington, 1975) was performed
using at a significance level of P ¼ 0.05.
into winter, probably because of nitrification of
accumulated NHþ
4 as well as catchment inputs.
Molar TN : TP and DIN : TP ratios (Fig. 1c) were
relatively high during late autumn and winter (83–
151 : 1 and 19–75 : 1, respectively) but, in summer, fell
below the thresholds for N limitation suggested by
Morris & Lewis (1988), 33 : 1 and 3 : 1 respectively.
TN : TP ranged from 24–34 : 1, while DIN : TP was
normally between 0.2–0.5 : 1 (except on 29 June when
it rose to 10 : 1). Throughout the growth season,
TN : TP ratio was no more than half the threshold
level above which Smith (1983) predicts cyanobacterial scarcity.
Results
Physicochemical conditions
Shelburne Pond displayed many symptoms of eutrophy during our study, including a TP concentration of
0.9–7.0 lM (0.1–0.4 lM present as SRP) and a TN
concentration of 66–137 lM (Fig. 1a,b). Ammonium
and nitrate concentrations were substantial (26.7 and
1.6 lM ) when the study began in May, but were
greatly reduced (to 1.45 and 0.01 lM ) by the first
cyanobacterial bloom in June (Fig. 1b). Decomposition
of this bloom later in the month may have been
responsible for the pulse in NHþ
4 (34 lM ) observed on
29 June; otherwise both fractions were present at very
low concentrations (NHþ
4 , 0.7–1.6 lM ; NO3 , 0.01–
0.05 lM ) until late August. Ammonium concentration
reached 31 lM on 4 September, and remained above
23 lM for the remainder of the growth season. Nitrate
concentration rose gradually from mid-September
Fig. 1 Phosphorus (a) and nitrogen (b) concentrations, and
molar N : P ratios (c) in Shelburne Pond, VT, between May 1999
and March 2000.
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
Cyanobacterial dominance and nitrogen fixation 697
The lake was circumneutral (pH 7.5–7.8) in winter
but during the growth season slightly alkaline (pH
8.2–9.2) (Fig. 2a). Consequently most of the DIC
available to phytoplankton was bicarbonate and
carbonate rather than CO2. DIC concentration over
the growth season was relatively stable, 2.32–
2.62 mM , but free CO2 varied widely with shifts in
temperature and pH (from 0.01 to 0.31 mM ; Fig. 2a).
However, free CO2 concentration did not fall below
the compensation concentration of chlorophytes
(approximately 0.01 mM , Shapiro, 1990).
Light extinction coefficient was high (1.12–3.80 m)1)
and Secchi depth low (0.40–1.68 m; Fig. 2b) in summer because of high algal turbidity. Although the lake
was frozen in winter and cool in early spring and late
autumn, temperatures of 21–28 C were maintained
from late May to mid-September (Fig. 2b).
Biological conditions and nitrogen demand
Chlorophyll a concentration was relatively low in May
(5–9 lg L)1) but rose to 60 lg L)1 by mid-June, and
for the remainder of the summer stayed above
(a)
(b)
d
Fig. 2 Free CO2 concentration (a), pH (a), temperature (b), and
Secchi depth (b) in Shelburne Pond, VT, between May 1999 and
March 2000.
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
30 lg L)1 (Fig. 3a). Blooms occurred in mid-June,
from mid-July to mid-August, and in mid-September,
yielding chlorophyll a concentrations of 60, 88–92 and
69 lg L)1, respectively. Surface scums were visible
during calm weather throughout most of the ice-free
season.
Diatoms dominated the phytoplankton community
from late autumn through spring but were replaced
by cyanobacteria in May (Fig. 3b). Aphanizomenon flosaquae, a nitrogen fixer, was the most abundant species
in the lake in early and late summer, but was
overwhelmed numerically by non-heterocystous P.
agardhii during much of July and August. Microcystis
aeruginosa also was present in appreciable numbers
during the period of P. agardhii dominance, and
Anabaena species (including A. flos-aquae, A. circinalis
and A. planctonica) were found in samples throughout
the ice-free season and for two months under ice.
Although Aphanizomenon numbers increased in autumn, following loss of P. agardhii from the lake,
Anabaena species were more important fixers during
this period. Heterocyst densities were consistently
low for the amount of Aphanizomenon and Anabaena
present. We measured 809 mL)1 in mid-June, but
<200 mL)1 during the remainder of the growth season
(Fig. 3a). The average number of heterocysts present
per 100 fixer cells was never greater than three
(Table 1).
Zooplankton grazers were most abundant in
spring, declined to a late summer minimum, and
then reached a smaller autumn peak. Rotifers made
up much of the community in spring, whereas
cladocerans were particularly important in summer.
Daphnia galeata mendotae was the most abundant
cladoceran, but was outnumbered by Chydorus
sphaeridus for a few weeks in midsummer. Copepods (especially Acanthocyclops varians and Diaptomus minuta) were present throughout the ice-free
season as subdominants.
Light response curves for primary productivity on
20 August indicated that carbon fixation saturated at a
light intensity of 200 lEi m)2 s)1 (Ik; Table 2). The
mean daytime light intensity in the mixed layer of the
lake (upper 1 m) was only about one third as high,
67 lEi m)2 s)1; consequently, primary productivity
was largely light limited at this time. Our primary
production model indicated that 370 kmol C were
fixed in the epilimnion during the 5 day period preceding our 20 August NSID sampling. Consequently,
698
L.R. Ferber et al.
(a)
(b)
Fig. 3 Heterocyst density (a), chlorophyll
a concentration (a), and the biovolume of
major phytoplankton groups in Shelburne
Pond, VT, between May 1999 and March
2000. Diatoms accounted for >95% of
chrysophyte biomass, except in January,
when Synura sp. made up approximately
40%.
Table 1. Measures of the importance of fixers to phytoplankton biomass, of heterocysts to cynobacterial cell counts, and of nitrogen
fixation to phytoplankton N nutrition. FC indicates fractional contribution; ISI, results based on tracer and acetylene reduction
studies conducted in the lake; and NSID, results obtained through measurement of natural stable isotope distribution. See text for
details. AT indicates that substrate concentrations were above the threshold value for N limitation (5 lM ); we did not use the mixing
model under these conditions
Date
(1999)
Fixer contribution
to biomass %
Heterocysts per
100 fixer cells
5/31
6/14
6/29
7/13
7/25
8/10
8/20
9/4
9/19
10/5
10/19
26.7
92.1
73.4
82.5
1.1
9.5
0.4
40.3
65.7
6.0
1.7
0.1
1.0
0.2
0.2
2.7
0.1
0.4
0.1
0.2
0.8
0.6
ISI estimates of FC
NSID estimates of FC
% N2
% NH4
% NO3
% N2
% DIN
–
–
0.2
–
1.8
–
0.7
–
0.0
–
0.2
83.3*
–
95.2
–
94.7
–
98.2
–
84.9
–
82.1
16.7*
–
4.6
–
3.4
–
1.1
–
15.1
–
17.7
AT
0.0
AT
0.0
8.8
0.0
0.0
0.0
AT
AT
AT
AT
100.0
AT
100.0
91.2
100.0
100.0
100.0
AT
AT
AT
*Percentage assumes no N2 fixation.
N demand, the uptake necessary to maintain the
molar C : N ratio of the phytoplankton (5.8), was
estimated as 64 kmol. This was the value for total CO2
fixation, including that associated with chemoauto-
trophy; with dark uptake correction, the estimate was
51 kmol. The phytoplankton N demand estimated
from phytoplankton biomass increased between 10
and 19 August was 36 kmol. The last value may be the
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
Cyanobacterial dominance and nitrogen fixation 699
Table 2 Parameters of light response curves for N2 and CO2 fixation, and for Michaelis–Menten uptake-subtrate relationships. a is the
slope of the rising limb of the light-response curves; Ns and Ps, the N2 and C fixation rates at light saturation; D, the N2 or C fixation
rate in the dark, and Ik, the light intensity at which N2 or C fixation saturates. Vmax is the uptake rate of NH4 or NH3 at substrate
saturation, and Km the half-saturation constant for uptake. Iave is the average light intensity of the epilimnion (assumed 1 m deep).
Light values are given both in the hourly units used in modelling, and the more familiar instantaneous units, lEi m)2 s)1 (in
parentheses)
Date
(1999)
a nmol N2 fixed
(106 heterocysts))1
(Ei m)2))1
Light response of N2 fixation
7/3
102
7/28
205
8/19
791
10/19
142
Date
(1999)
Ns
nmol N2 fixed
(106 heterocysts))1 h)1
D
nmol N2 fixed
(106 heterocysts))1 h)1
Ik
Ei m)2 h)1
(l Ei m)2 s)1)
r2
Iave
(lEi m)2 s)1)
Ei m)2 h)1
33
257
366
451
8.3
137
58
0
0.33
1.25
0.46
3.17
0.90
0.97
1.00
0.76
0.48
0.37
0.24
0.13
a nmol C fixed
(mg chl a))1
(Ei m)2))1
Light response of primary productivity
8/20
0.42
Ps
nmol C fixed
(mg chl a))1 h)1
Ik
Ei m)2 h)1
(lEi m)2 s)1)
21
0.72 (200)
(91)
(347)
(128)
(881)
r2
(133)
(103)
(67)
(36)
Iave
Ei m)2 h)1
(lEi m)2 s)1)
0.24 (67)
Date (1999)
Substrate
Vmax l mol N L)1 h)1
Km lM
r2
Michaelis–Menten parameters
8/19
8/19
NH4
NO3
3.00
0.65
1.68
0.50
0.68
1.00
most accurate as it takes into account respiration by
night. On the other hand, it does not account for
phytoplankton production lost to grazers.
Nitrogen fixation
In situ incubation of integrated epilimnetic samples
over depth profiles that exposed fixers to a light
gradient normally indicated significant surface inhibition, maximum fixation at 0.25 m, and a steep loglinear decrease in rates down to the lowest incubation
depth, 2 m. Fitting of the light response equations of
Lewis & Levine (1984) to the data yielded low r2
values (<0.5) when a surface inhibition term was
included, but r2 values in excess of 0.9 when it was
not. Because Bower et al. (1987) have shown that
freely circulating (and migrating) algal communities
do not experience the surface inhibition of photosynthesis that occurs in bottles held artificially at the
surface and subject to overheating and photo-oxidation, we decided to use the higher r2 equations
without surface inhibition in our numerical model.
These equations indicated light-saturated fixation
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
rates of 33–451 nmol (million heterocysts))1 h)1, and
Ik values of 36–133 lEi m)2 s)1 (Table 2), results
comparable with those obtained in previous studies
of the light response of N2 fixation (Lewis & Levine,
1984; Mugidde et al., 2004). Fixation saturated at a
lower light intensity and achieved a lower maximum
velocity in summer, when A. flos-aquae was the
principal fixer, than in autumn when Anabaena spp.
were more important. During four of five samplings,
the average light intensity in the epilimnion (Table 2)
was below estimated Ik values, suggesting light
limitation of nitrogen fixation.
Mean ambient nitrogen fixation rates in the epilimnion (for the 5-day estimation periods) ranged from
0.4 to 9.6 nmol L)1 h)1 (Table 3). We do not include
the data for 19 September in the above ranges, as
2 days before this sampling Vermont’s largest storm
of the decade passed over Shelburne Pond, mixing it
to the bottom and depositing 26 cm of rain. The
bloom-forming cyanobacteria are notoriously sensitive to weather changes, especially turbulent mixing,
which may break up filaments (Paerl et al., 1981;
Moisander et al., 2002). That we measured no fixation
700
L.R. Ferber et al.
Date (1999)
6/3
NH+4 uptake
DT, lmol L)1 h)1
DT, lmol lg chl a)1 h)1
MM, lmol L)1 h)1
MM, lmol lg chl a)1 h)1
NO)3 uptake
DT, lmol L)1 h)1
DT, lmol lg chl a)1 h)1
MM, lmol L)1 h)1
MM, lmol lg chl a)1 h)1
N2 fixation
nmol L)1 h)1
nmol lg chl a)1 h)1
nmol (million hetero cysts))1 h)1
Total N incorporation
kmol (N epilimnion))1 5 (d))1
Turnover times
NH+4, h
NO)3, h
Particulate N, h
12.3
2.01
0.4
0.07
0.34
0.06
0.08
0.013
–
–
–
6/29
2.6
0.11
1.87
0.08
0.05
0.00
0.09
0.004
0.8
0.025
10.3
7/25
8/20
40.2
0.62
2.48
0.04
3.26
0.05
0.09
0.001
9/18
10/19
2.7
0.08
0.89
0.03
4.3
0.09
3.94
0.08
3.4
0.43
0.65
0.08
0.4
0.01
0.01
0.000
0.04
0.00
0.7
0.014
0.01
0.00
0.14
0.018
9.62
1.28
0.106
0.026
52
106.6
0
0
0
0.38
0.01
13.2
21.7
88.7
118.2
40.9
209.5
35.8
13.8
15.4
83.2
17.9
1.6
27.8
0.1
0.4
40.1
0.8
1.0
95.9
5.9
2.3
21.9
47.2
51.3
73.7
on 19 September despite substantial fixer presence
may thus be related to disturbance.
Dissolved inorganic nitrogen uptake
The Michaelis–Menten analyses performed on 19
August (Table 2) revealed ambient nitrate and ammonium uptake velocities well below those at substrate
saturation (Vmax). The half saturation constant (Km) of
þ
NHþ
4 uptake was similar to NH4 concentrations in
summer, whereas the value for nitrate uptake,
0.50 lM , was 10–50 times greater than the amount of
substrate present (suggesting poor access to this N
form).
Michaelis–Menten and direct tracer estimates of
ammonium uptake were in agreement on 29 June and
18 September, when substrate concentrations were
sufficient to saturate uptake (Table 3). At other times
during the growth season, the MM rate was substantially lower, indicating that substrate limitation
occurred not just in August, but was the general rule.
Volumetric ammonium uptake rate ranged from 0.40
to 3.94 lmol L)1 h)1. Because nitrate concentration
was very low in summer, uptake rate for this substrate
was only 0.01–0.09 lmol L)1 h)1 (Table 3). The rise in
nitrate concentration in autumn stimulated uptake by
more than 10-fold. The turnover times, calculated by
Table 3 Average rates of NH+4 and NO)3
uptake [as estimated from direct tracer
(DT) and Michaelis–Menton (MM) techniques] and N2 fixation in the epilimnion
(upper 1 m) of Shelburne Pond. Total
inorganic N incorporation over 5-day
periods (ending on stated dates) and
pool turnover times also are given (using
Michaelis–Menton estimates of uptake
rates and assuming no dissolved organic
nitrogen use by phytoplankton)
)
dividing the measured NHþ
4 and NO 3 concentrations
by uptake rates, were consistently less than a day, and
in mid-summer fell to <1 h (Table 3).
Light intensity had little impact on either NHþ
4 or
NO)3 uptake rates in the lake (Fig. 4). One-way
A N O V A and Student–Newman–Keul tests indicated
that the rates measured at different light intensities
(4.1–4.5 lmol N L)1 h)1 for NHþ
and 0.35–
4,
0.44 lmol N L)1 h)1 for NO)3) were not significantly
different from one another (P ¼ 0.05).
Fig. 4 Direct tracer (DT) ammonium and nitrate uptake rates at
different light intensities.
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
Cyanobacterial dominance and nitrogen fixation 701
Fractional contributions estimated from in-situ
incubations
Comparison of the 5-day estimates of NO)3 , NHþ
4 and
N2 use over the depth of the epilimnion (Table 1)
indicated consistent reliance on NHþ
4 ; 82–98% of the
inorganic N used was NHþ
.
Nitrate
uptake provided
4
15–18% of the inorganic N assimilated in early
summer and autumn, but only 1–5% in July and
August. Nitrogen fixation was the smallest source,
contributing from 0 to 2%. Total inorganic N incorporation over the 5-day integration periods ranged
from 22 to 210 kmol (Table 3). For 16–20 August, the
period for which we have an estimate of phytoplankton N demand, the value was 41 kmol. Particulate N
turnover time, estimated by dividing PN by total N
incorporation rate, ranged from 1 to 4 days, and had
no obvious temporal pattern (Table 3).
Natural stable isotope distribution (d15N)
Little data exist on the d15N signatures of freshwater
phytoplankton, and even less on those of DIN. Our
study revealed seasonal patterns for both parameters.
Ammonium d15N signature (Fig. 5) was high (9–24&)
in spring and again in autumn and winter, but
plunged to values of )0.2–2.4& in summer. Nitrate
d15N signature could not be measured in summer
because of extremely low nitrate concentration. For
late autumn and winter, the signature ranged from 6.7
to 9.1&, while in late spring and early autumn, it was
a little lower, 4.2–5.6&. Nitrate signature was consistently lower than NHþ
4 signature (on five occasions,
by 14–15&). The average standard deviation of
duplicates was substantial for both analyses, 2.2&
for nitrate and 2.5& for ammonium (but 1.7& for the
summer time period in which we used the mixing
model). Nevertheless, t-tests indicated that the central
tendency for both signatures was significantly different from )2& (P < 0.0001 for spring & autumn;
<0.025 for midsummer). Thus, one of the prerequisites
of model use was met.
Phytoplankton signature was less variable than that
of NHþ
4 but still displayed a distinct seasonal pattern
(Fig. 5). Values of 5.2–9.4& were obtained in winter,
followed by negative values in spring, a plateau at 2–
3& in July and August, and negative values again in
autumn (with a temporary rise back to +2.5& following a storm in September). The standard deviation of
duplicates was low, 0.1& on average. Phytoplankton
) 15
d15N was much lighter than NHþ
4 and NO 3d N
during spring, autumn and winter. However, in
summer the phytoplankton and NHþ
4 signatures were
similar, or slightly more positive.
There were five sampling dates in summer when we
were able to use the mixing model to estimate relative
source contributions to phytoplankton N nutrition
(dates when DIN concentration was <5 lM ). On one
of these, we estimated a 91% contribution to phytoplankton nutrition from DIN, and a 9% contribution
from nitrogen fixation (Table 1). For the other four, the
model suggested that 100% of the N was from NHþ
4 . In
fact, phytoplankton signatures were slightly more
positive than those of NHþ
4 , so that the model predictions were negative percentages, which were arbitrarily
assigned values of 0%. High sample standard deviation
or a systematic error in our DIN measurement may
have caused the DIN values to fall below the values of
phytoplankton and create the negative values. An
alternative hypothesis is that phytoplankton took up
some N from an unmeasured source with a more
positive signature, e.g. dissolved organic nitrogen and/
or NHþ
4 diffusing from sediments.
Correlations
Fig. 5 The d15N signatures of phytoplankton, NHþ
4 , and NO3 in
Shelburne Pond, VT, between May 1999 and March 2000. The
theoretical signature of cyanobacteria using only N2 ()2&) also
is given.
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
Pearson product-moment correlation analysis indicated that algal biomass played a major role in determining the physicochemical characteristics of
Shelburne Pond. Secchi depth, DIC, DIN (both NHþ
4
and NO)3) and molar TN : TP and DIN : TP ratios all
fell with rising chlorophyll a concentration, while pH
702
L.R. Ferber et al.
Independent variables
Dependent variable
Positively correlated
Negatively correlated
Chlorophyll a
TP*, TN*, temperature*, pH
% Cyanobacteria
% N2 fixers
Cyanobacterial biomass
NC
NC
Chlorophyll a*, TP*, TN,
pH, temperature
Chlorophyll a*, TP*, TN
Chlorophyll a, heterocysts
TN : TP
TN : TP*, DIN : TP*,
DIN*, Secchi depth, DIC
NC
NC
NO
3 , NH4 , TN : TP, DIN :
TP Secchi depth, DIC
NO
3 , TN : TP, DIN : TP, DIC
NC
N2 fixer biomass
N2 fixation rate
N source: % NO
3
% NHþ
4
Phytoplankton d15N
Table 4 Relationships between phytoplankton and environmental variables
found to be significant at P < 0.05 in
Pearson Product moment correlation
analysis
TN : TP
TN : TP
*Significance at P < 0.05.
NC, no correlations.
increased (Table 4). Statistically significant correlations of chlorophyll a with TP and TN suggested that
both nutrients regulated algal biomass, while a negative correlation with TN : TP and DIN : TP ratios
indicated more pressure on N supplies. The relative
contribution of cyanobacteria to phytoplankton biomass did not correlate with any measured environmental variable, probably because the group
dominated throughout the ice-free season (and over
all conditions). Separate analysis of the relative contribution of nitrogen fixers also showed no correlations. However, cyanobacterial and fixer biomass
correlated with most of the variables that influenced
chlorophyll a biomass, although usually at a lower
level of significance.
We had few data points to seek relationships
between N-use rates and physiochemical parameters.
Nitrogen fixation rate correlated with neither DIN
concentration nor N : P ratios, but was related to
heterocyst density and chlorophyll a. The relative
contribution of NO)3 to N nutrition increased with
TN : TP ratio, while that of NHþ
4 decreased. Negative
correlations between phytoplankton d15N signatures
and chlorophyll a, heterocyst abundance and cyanobacterial relative abundance were expected, but not
found. The former correlated only with TN : TP.
Discussion
Validity of the low N : P hypothesis
The principal objective of this study was to determine
whether the vacuolated cyanobacteria that form
surface scums and dominate the biomass of many
eutrophic lakes rely on atmospheric nitrogen as their
primary N source. This situation must be the case if
the popular low N : P hypothesis is generally true, as
its fundamental premise is that these algae overcome
N shortage through fixation while other species are
halted in their growth. Using two separate methods to
estimate the relative contributions of DIN and atmospheric nitrogen to phytoplankton nutrition in bloomprone Shelburne Pond during 1999, we found that
fixation rarely supplied more than 2% of the N
required. Rather, ammonium was the principal N
source. This was true not only during periods when
eukaryotic algae or nonheterocystous cyanobacteria
such as Planktothrix and Microcystis dominated, but
also during June and September when Aphanizomenon
and Anabaena accounted for 73–92 and 40–66% of
phytoplankton biomass, respectively (Table 1). Low
reliance of Aphanizomenon and Anabaena on nitrogen
fixation also was implied by heterocysts counts.
Heterocyst density rarely exceeded 200 L)1, and the
heterocyst frequency on fixer filaments consistently
was <3%. By contrast, heterocyst densities >2000 L)1
and heterocyst frequencies of up to 24% have been
reported for lakes with vigorous nitrogen fixation
(Levine & Lewis, 1987; Findlay et al., 1994).
Without a long-term record, we cannot say whether
the low fixation rates observed in 1999 are normal for
Shelburne Pond. Lescaze (1999) included Shelburne
Pond in a cross-lake comparison of N stable isotope
chemistry undertaken in 1997. Incorporation of her
data into our mixing model suggests that nitrogen
fixation accounted for about 28% of phytoplankton N
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
Cyanobacterial dominance and nitrogen fixation 703
nutrition during 1997. Higher fixation rates were
associated with DIN concentrations that were an
order of magnitude lower than in 1999. Ironically,
DIN concentrations were lower because the year was
wetter and more N and P were brought into the lake.
The extra nutrients allowed for more algal growth,
which brought down not only the new pools of
nutrient, but also those initially present. Heterocyst
densities were three times greater in 1997 than in 1999,
attesting to greater dependency on fixation.
It appears then that N use dynamics vary annually,
depending in part on the vagrancies of weather. In
some years, nitrogen fixation may be necessary to
support high biomass; in other years, when biomass is
lower, ammonium recycling from zooplankton, microbes and the sediments may be sufficient. In any
case, we found fixers present and even dominant
during periods of low nitrogen fixation, and must
conclude that while fixation may be one means of
heterocystous cyanobacteria outcompeting other algae, it is not the only one.
Relevance of the low DIN Hypotheses
Many indicators of N stress were apparent in Shelburne
Pond during the summer and autumn bloom periods.
DIN concentration was normally <1 lM; TN : TP and
DIN : TP ratios were less than the threshold values for
N limitation (33 and 3, respectively) suggested by
Morris & Lewis (1988); NHþ
4 and NO3 uptake rates
were well below Vmax; and negligible isotopic fractionation occurred during DIN uptake. The low DIN
hypothesis predicts different cyanobacterial dominants
during N limitation, depending on the sources of N
available. Picoplanktonic species are expected when
the principal source is ammonium recycled within the
water column; colonial and vacuolated nonheterocystous species such as Microcystis, Oscillatoria and Planktothrix when a benthic ammonium source can be
reached through vertical migration; and heterocystous
species when neither of these sources is significant and
nitrogen fixation must be relied upon (Blomqvist et al.,
1994; Hyenstrand et al., 1998a,b). Picoplanktonic cynaobacteria were scarce in Shelburne Pond during 1999,
but Microcystis and Planktotrix were major genera in
summer. Conditions in the lake are appropriate for
exploitation of a benthic N source. Mean depth is just
3.4 m, a short distance for vacuolated cyanobacteria to
migrate to the bed yet still be able to spend much of the
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
day photosynthesizing near the lake surface. There are
no data on NHþ
4 flux from sediments, but the sediments
probably are N-rich, the lake having been hypertrophic
for decades. That we measured a shortfall of 35% in N
uptake relative to N demand in the epilimnion in
August while nonheterocystous vacuolated cyanobacteria dominated is consistent with the thesis. On the
other hand, part of the shortfall may have been met by
use of dissolved organic N. We did not have the
resources to estimate uptake rates for the many possible
organic substrates in the lake. It is also a distinct
possibility that Anabaena and Aphanizomenon also may
have met some of their N demand through vertical
migrations into the hypolimnion or to the sediments,
and thus avoided the need to fix nitrogen. The strong
migratory abilities of these genera are well documented
(Reynolds, 1984), as is their preference for NHþ
4 over N2
(Raven, Evert & Eichhorn, 1992). We recommend that
the low DIN hypothesis be modified to allow both
nonheterocystous and heterocystous vacuolated cyanobacteria the opportunity to outcompete other species
through migrations that give them superior access to
nutrient sources, P as well as N.
Another prediction of the low DIN hypothesis
supported by this study relates to eukaryote dominance during episodes of N scarcity. The hypothesis
predicts dominance by eukaryotes when the principal
form of N available is nitrate (more specifically it
requires that nitrate concentration exceed 1.4 lM ;
Blomqvist et al., 1994). The basis of the prediction is
the more efficient nitrate reductase in eukaryotes than
prokaryotes. In Shelburne Pond, diatoms dominated
from late autumn until spring, the period when nitrate
was available at concentrations >1.4 lM . The observed
succession pattern also was consistent with McQueen
& Lean’s (1987) earlier prediction of eukaryote dominance at NO
3 : TP ratios >11 : 1 and temperatures
<21 C. Caution must be exercised in interpreting
empirical relationships, however. Many physicochemical attributes of Shelburne Pond change with season,
making indirect and spurious relationships a strong
possibility. The role of nitrate in prompting successions to eukaryotes requires further analysis.
Considerations of C and light based hypotheses
Although Shapiro (1990) has argued strongly that CO2
shortages are more important than N shortages in
favoring cyanobacteria, we found no evidence that C
704
L.R. Ferber et al.
was limiting in Shelburne Pond during 1999. Because
of dolomite in its catchment, the lake has exceptionally high DIC concentrations. Consequently, even at
the greatest pH measured, 9.2, equilibrium levels of
CO2 exceeded the compensation concentrations for
chlorophytes reported by Shapiro (1990). Correlation
analysis showed weak negative relationships between
cyanobacterial biomass and DIC and CO2 concentration.
Arguments for light limitation as a factor favouring
cyanobacteria focus on the ability of these algae to
concentrate at the lake surface where light availability
is greatest and, in so doing, shade other species
(Zevenboom & Mur, 1980; Présing et al., 1999). In
addition, cyanobacteria appear to have lower Ik values
for photosynthesis than eukaryotes (Présing et al.,
1999) and thus may be better competitors even when
mixing is sufficient to prevent buoyancy regulation. A
negative relationship between cyanobacterial biomass
and Secchi depth was obtained for this study, but we
did not find statistically significant relationships
between the relative abundance of either fixers or
non-fixing cyanobacteria and more direct measures of
light availability. However, our limited estimates of Ik
values for fixation and primary production were
greater than the mean daily light availability in the
mixed layer, suggesting occasional light limitation of
both processes.
The generality of our results
There have been relatively few simultaneous measurements of nitrogen fixation and DIN uptake in lakes
(Billaud, 1968; Takahashi & Saijo, 1988; Gu &
)
Alexander, 1993b). Comparisons of NHþ
4 and NO 3
uptake rates have been more common, and consistently have shown that NHþ
4 is the dominant N source
on an annual basis (e.g. Brezonik, 1972; Liao & Lean,
1978a,b; Axler, Redfield & Goldman, 1981; Berman
et al., 1984; Fisher et al., 1988). Only occasionally, in
spring or autumn, do phytoplankton use as much or
more NO)3 (Billaud, 1968; Berman et al., 1984;
Takahashi & Saijo, 1988). When nitrogen fixation has
been included in source assessment, its contribution
to phytoplankton nutrition generally falls behind
those of nitrate and urea (Billaud, 1968; Takahashi &
Saijo, 1988; Gu & Alexander, 1993a; Présing et al.,
1999). Occasionally major blooms bring in as much as
50–75% of the total N demand via fixation (Gu &
Alexander, 1993a,b), but most studies indicate summer contributions of <10% (Billaud, 1968; Takahashi
& Saijo, 1988; Présing et al., 1999).
Howarth et al.’s (1988) frequently referenced review
of nitrogen fixation in lakes compares quantities of N
fixed with allochthonous inputs, rather than with use
of internal sources. Because many lakes receive little
N from the catchment in summer, but recycle large
amounts of NHþ
4 , an exaggerated impression of the
role of nitrogen fixation in a lake’s N dynamics may
emerge. For example, Mugidde et al. (2004) found that
nitrogen fixation accounted for approximately 80% of
the total annual N input to Lake Victoria (external
plus fixation), but supplied just 2% on average of the
N incorporated into phytoplankton daily. Several
studies have demonstrated that rates of zooplankton
N excretion and N remineralization from detritus in
lakes are generally sufficient to support algal growth
(e.g. Liao & Lean, 1978b; Axler et al., 1981; Morrissey
& Fisher, 1988).
A separate issue from the dynamics of competition
is whether nitrogen fixation succeeds in maintaining P
limitation in lakes undergoing eutrophication, as
Schindler (1977) predicted it would. Although the
atmospheric supply of N2 places no limit on the
amount of N that can be brought into a lake through
fixation, most cyanobacterial fixers rely heavily on
photoreactions to meet the high ATP demand of
fixation. Consequently, fixation can be limited by
light. In the small lakes Schindler studied, epilimnia
were shallow and sufficient light was available for
fixation to keep pace with P increases. In larger lakes
with deeper mixed layers, average light availability
can be low enough to constrain nitrogen fixation and
result in symptoms of N limitation among heterocystous cyanobacteria as well as non-fixers (Levine &
Lewis, 1987; Mugidde et al., 2004). The present study
indicated that low light also might prevent compensation for N shortages through fixation in shallow
lakes, when surface scums greatly reduce light
extinction rates. The mixed layer in Shelburne Pond
was just 1 m deep during stratification, but nitrogen
fixation was light limited below 0.5 m. Calm weather
allowed fixers to position themselves near the lake
surface, but windy weather mixed them into the zone
of light limitation. This situation is probably not
unusual. In a review of nutrient limitation studies in
North American lakes, Elser, Marzolf & Goldman
(1990) concluded that despite a common belief to the
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
Cyanobacterial dominance and nitrogen fixation 705
contrary, N limitation is almost as common in lakes as
P limitation.
Methodological issues
While heterocyst and cell counts are straightforward,
and the determination of heterocyst frequency a
simple matter of division, extrapolation of the results
of the ISI and NSID assays to the lake involved
modelling with assumptions that influenced results.
In the case of the ISI assay, all major assumptions (that
þ
MM estimates of NO
3 and NH4 uptake reflected
ambient rates better than the higher DT estimates; that
nitrogen fixation was stimulated by light while DIN
uptake was not; and that the epilimnion was consistently shallow enough (1 m) to keep phytoplankton in
a lighted region) resulted in a bias toward overestimation of fixation relative to DIN uptake. Despite this
fact, we found ammonium uptake to be the principal
N source for phytoplankton.
The NSID analysis naturally integrates over epilimnion volume and phytoplankton lifetimes making
sampling and modeling easy. Because it involves so
little extrapolation, it should provide more reliable
estimates than the ISI method. However, its outcome
is highly sensitive to the fractionation estimates and
d15N values of phytoplankton and substrate. We have
considerable confidence in the accuracy of our phytoplankton data, the replication of samples being good
and the values obtained similar to those measured in
other lakes with cyanobacterial dominants (A. Lini
and S. Levine, unpublished data). The fractionation
values for N2 and NHþ
4 use have been obtained
repeatedly in studies of N-limited phytoplankton in
culture using these substrates exclusively; we have
confidence in them as well. Any error in the method is
probably because of variability in our estimates of the
d15N of ammonium. Duplicate samples frequently
differed by 20–30%, and analysis of standards indicated substantial isotopic fractionation during extraction and concentration, which we corrected for in our
modelling. This fractionation might be related to
contamination with atmospheric ammonia or microbial growth, as sample processing requires several
days. If so, variability amongst replicates could be
related to unequal contamination. Improvements in
this method are required before it can be widely used.
NSID results are included here because we feel other
researchers should be aware of the method’s potential
2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708
power as well as its current weaknesses, and because
the data we obtained were consistent with the
findings of our other two assays.
In conclusion, effective management of cyanobacterial blooms requires understanding of the circumstances that allow bloom-forming species to dominate.
The low N : P hypothesis suggests one situation, DIN
availability so low that only nitrogen fixers are able to
obtain enough N to sustain and increase growth. Just as
checkmate in chess may be achieved through more than
one series of plays, we believe that fixation may be just
one means by which cyanobacteria may dominate the
phytoplankton during eutrophication. Vertical migration to benthic ammonium sources is another possible
mechanism, as is dissolved organic N (DON) use, or the
formation of surface scums that induce light limitation
in subsurface populations and thus reduce competition
for DIN. Some of these alternative mechanisms are also
available to nonheterocystous cyanobacteria, which
may explain why mixtures of the two groups are
common in many eutrophic lakes. We do not reject the
low N : P hypothesis outright but suggest that the
situation is even more interesting than the scenario put
forth by Schindler (1977).
Acknowledgments
This project was funded through grants from the U.S.
Geological Survey (HQ96GR02702), the Lintilhac
Foundation and the Lake Champlain Research Consortium. B. Buckley (University of Rhode Island
School of Oceanography) and N. Kamman (Vermont
Agency of Natural Resources) performed analyses of
dissolved and total nutrients, respectively, and
R. Michener (Boston University) developed and
helped us carry out the d15N-DIN analyses. We thank
B. Rosen, M. Lescaze, A. Shambaugh, A. Howard,
H. Hales, B. Hunt, and others for additional advice
and technical assistance. J. Lehman provided useful
criticism of an early draft.
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