Do cyanobacteria dominate in eutrophic lakes because they fix atmospheric nitrogen?

Do cyanobacteria dominate in eutrophic lakes becausethey 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.

SUMMARY

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, stableisotope

Introduction

Cyanobacterial blooms are a frequent and unwelcome

consequence of lake eutrophication. With rising nutri-

ent 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.

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 cyano-

bacteria from a poorly- to a highly-competitive posi-

tion. 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 hypothe-

ses suggest that, once all resource limitations are

Correspondence: S. N. Levine, Rubenstein School of

Environment and Natural Resources, University of Vermont,

Burlington, VT 05405, U.S.A. E-mail: [email protected]

Freshwater Biology (2004) 49, 690–708

690 � 2004 Blackwell Publishing Ltd

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 sub-

stantially reduced by vacuolation (Knoechel & Kalff,

1975).

Here we provide a test of the most widely accepted

hypothesis about cyanobacterial dominance in eu-

trophic 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 cyanobac-

teria 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 unusu-

ally 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 phys-

iology to provide a convincing explanation. He specu-

lated 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 sup-

plies 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 Aphani-

zomenon occurred when the enrichment ratio was

lower. Smith (1983) further promoted the hypothesis

by showing that the relative abundance of cyanobac-

teria in 17 north temperate lakes correlated negatively

with total N : total P (TN : TP) ratio.

Previous tests of the low N : P hypothesis contin-

ued the field enrichment studies and empirical ana-

lyses initiated by Schindler (1977) and Smith (1983),

but with less straightforward results. Nitrogen addi-

tions 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) cyanobac-

teria, 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-fix-

ing 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 cyanobac-

terial taxa (Oscillatoria, Pseudoanabaena and Lyngbya)

increased in relative abundance along an N : P gradi-

ent. 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, Willen &

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 heterocys-

tous versus nonheterocystous cyanobacterial domin-

ance 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-

Cyanobacterial dominance and nitrogen fixation 691

� 2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 690–708

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 hyper-

trophic lake over the course of an ice-free season,

looking for a relationship between periods of hetero-

cystous 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 eval-

uation 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.

Methods

Study site

Shelburne Pond, 16 km southeast of Burlington, VT,

U.S.A. (44�23¢N, 73�9.5¢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 sum-

mer. 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 lMM, respectively; chlorophyll a

concentration, 88 lg L)1 ; and Secchi depth, 0.78 m

(N. Kamman, Vermont Department of Environmental

Conservation, pers. comm.). The mean TN concentra-

tion for 1997–99 was 124 lMM (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 domin-

ated 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) Brebisson, A. circinalis Rabenhorst, and A.

planctonica Brunnth.), or by non-heterocystous Plank-

tothrix agardhii (Komarek) Anagnostides (probably

what DeYoe (1981) called Oscillatoria subbrevis Schm-

idle) 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 Chydo-

rus sphaerida O.F. Muller), copepods (mostly Acantho-

cyclops 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 (Notemig-

onus crysoleucas Mitchell) are the most abundant fish

(B. Chipman, Vermont Fish and Wildlife, pers. comm).

Nitrogen fixation

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) with15N2, determining the isotopic content of phytoplankton

692 L.R. Ferber et al.


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 light-

sensitive 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 I0e�ez; 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 deter-

mined using the equation:

Ni ¼ Ns � Nse�a þ D; ð2Þ

where a ¼ aIN�1s . 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 NHþ4 uptake rates generally were meas-

ured 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 lMM) or NaNO3 (0.13 lMM) and the bottles incuba-

ted 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 precom-

busted GF/C glass fibre filters. The filters were oven-

dried at 60 �C, sealed in quartz tubes containing CuO

(3.0 g), CaO (approximately 0.5 g), and Cu (2.5 g) and

combusted at 900 �C (Kendall & Grim, 1990). The

resulting nitrogen gas was analysed to determine its15N/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:

U ¼ dðatom%P15NÞdt

� PN

atom % 15NHþ4 or 15NO�

3

;

ð3Þ

where U ¼ uptake rate in lmol L)1 h)1, d(atom %

P15N) is the change of the isotopic composition of the

PN pool over time); PN is the size of the particulate

nitrogen pool; and atom per cent 15NHþ4 or 15NO�

3 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 addi-

tions, 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 concen-

trations 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. Nitro-

gen-sufficient algae should take up N near the maxi-

mum 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 equa-

tions 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 concentra-

tions (0, 0.07, 0.15, 0.36, 0.71 and 1.79 lMM) and

incubated at a depth of 0.33 m for 4–5 h. Rate

constants for uptake were obtained from the differ-

ence in particulate 15N content at the beginning and

end of the incubation period. To facilitate the use of



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:

Uca ¼ Uc

max

S

Km þ S� chl:; ð4Þ

where Uca is the ambient uptake rate (lmol L)1 h)1);

Ucmax is the maximum uptake velocity [lmol (lg

chlorophyll a L)1) h)1); S is the substrate concentration

(lMM); Km is the half-saturation constant (lMM); 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 dia-

toms dominant, so the equation was inappropriate.

Fortunately, substrate concentrations were high dur-

ing these periods, so that the direct tracer measures are

probably good estimators of ambient rates.

On 20 August, we examined the relationship

between NO�3 and NHþ

4 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-stimula-

ted 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 NHþ4 and NO�

3

generally differ from that of atmospheric nitrogen.

The phytoplankton signature reflects the ‘mix’ of

source use. Specifically,

PC ¼ðd15Ni � fiÞ � d15Np

ðd15Ni � fiÞ � ðd15Na � faÞ� 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 nitro-

gen fixation, respectively (modified from Shearer &

Kohl, 1989). Delta 15N is defined as

d15Nð&Þ ¼ð15N=14NÞsample � ð15N=14NÞstandard

ð15N=14NÞstandard

� 1000;

ð6Þ

where the standard is atmospheric nitrogen (the

signature of which is defined as 0&). d15Ni is

generally a composite variable, as NHþ4 and NO�

3

may have different isotopic signatures. However, we

use only the value of d15NHþ4 here because NHþ

4

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 fiis 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 lMM. We used a more conservative

threshold for the decision to assume no fractionation,

ammonium concentrations <5 lMM, plus other symp-

toms 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 distri-

bution analysis from the epilimnion of Shelburne

Pond biweekly during the ice-free season of 1999

(May to November), and then monthly from Decem-

ber 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



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 oven-

dried (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 extrac-

tion on thawed samples was according to the proce-

dure of Downs et al. (1999). Ammonium and nitrate

(2–15 lmol) were collected onto DOWEX-50W and

DOWEX-1 resins, respectively, by pumping or grav-

ity-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 MM 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 frac-

tionation 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, To-

kyo, Japan) at 400· magnification (Utermohl, 1958).

At least 100 fields or 100 heterocysts were counted

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 mea-

sured 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 precom-

busted GF/C filters in a CE Instruments C/N

analyser. These results did not take into account

phytoplankton respiratory losses at night. Conse-

quently, 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 Shim-

adzu 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



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 con-

centrations (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

NHþ4 was significantly different from )2&, the nor-

mal value for phytoplankton totally reliant on fixation

as a N source. One-way ANOVAANOVA using the Student–

Newman–Keuls (SNK) test was used to test for

differences in NHþ4 uptake rates during the light-

uptake experiment. To investigate the role of envi-

ronmental parameters on NHþ4 and NO�

3 uptake and

N2 fixation rates, and on natural stable isotope

distribution results, Pearson product moment corre-

lation analysis (Darlington, 1975) was performed

using at a significance level of P ¼ 0.05.

Results

Physicochemical conditions

Shelburne Pond displayed many symptoms of eutro-

phy during our study, including a TP concentration of

0.9–7.0 lMM (0.1–0.4 lMM present as SRP) and a TN

concentration of 66–137 lMM (Fig. 1a,b). Ammonium

and nitrate concentrations were substantial (26.7 and

1.6 lMM) when the study began in May, but were

greatly reduced (to 1.45 and 0.01 lMM) 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 NHþ4 (34 lMM) observed on

29 June; otherwise both fractions were present at very

low concentrations (NHþ4 , 0.7–1.6 lMM; NO�

3 , 0.01–

0.05 lMM) until late August. Ammonium concentration

reached 31 lMM on 4 September, and remained above

23 lMM for the remainder of the growth season. Nitrate

concentration rose gradually from mid-September

into winter, probably because of nitrification of

accumulated NHþ4 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 cyanobacte-

rial scarcity.

Fig. 1 Phosphorus (a) and nitrogen (b) concentrations, and

molar N : P ratios (c) in Shelburne Pond, VT, between May 1999

and March 2000.



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 mMM, but free CO2 varied widely with shifts in

temperature and pH (from 0.01 to 0.31 mMM; Fig. 2a).

However, free CO2 concentration did not fall below

the compensation concentration of chlorophytes

(approximately 0.01 mMM, 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 sum-

mer 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

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 flos-

aquae, 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 au-

tumn, 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. Cope-

pods (especially Acanthocyclops varians and Diapto-

mus 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 pre-

ceding our 20 August NSID sampling. Consequently,

(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.



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

(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 lMM); we did not use the mixing

model under these conditions

Date

(1999)

Fixer contribution

to biomass %

Heterocysts per

100 fixer cells

ISI estimates of FC NSID estimates of FC

% N2 % NH4 % NO3 % N2 % DIN

5/31 26.7 0.1 – 83.3* 16.7* AT AT

6/14 92.1 1.0 – – – 0.0 100.0

6/29 73.4 0.2 0.2 95.2 4.6 AT AT

7/13 82.5 0.2 – – – 0.0 100.0

7/25 1.1 2.7 1.8 94.7 3.4 8.8 91.2

8/10 9.5 0.1 – – – 0.0 100.0

8/20 0.4 0.4 0.7 98.2 1.1 0.0 100.0

9/4 40.3 0.1 – – – 0.0 100.0

9/19 65.7 0.2 0.0 84.9 15.1 AT AT

10/5 6.0 0.8 – – – AT AT

10/19 1.7 0.6 0.2 82.1 17.7 AT AT

*Percentage assumes no N2 fixation.



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 inhi-

bition, maximum fixation at 0.25 m, and a steep log-

linear 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 photosyn-

thesis that occurs in bottles held artificially at the

surface and subject to overheating and photo-oxida-

tion, we decided to use the higher r2 equations

without surface inhibition in our numerical model.

These equations indicated light-saturated fixation

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 epilim-

nion (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 sensi-

tive to weather changes, especially turbulent mixing,

which may break up filaments (Paerl et al., 1981;

Moisander et al., 2002). That we measured no fixation

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

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

Light response of N2 fixation

7/3 102 33 8.3 0.33 (91) 0.90 0.48 (133)

7/28 205 257 137 1.25 (347) 0.97 0.37 (103)

8/19 791 366 58 0.46 (128) 1.00 0.24 (67)

10/19 142 451 0 3.17 (881) 0.76 0.13 (36)

Date

(1999)

a nmol C fixed

(mg chl a))1

(Ei m)2))1

Ps

nmol C fixed

(mg chl a))1 h)1

Ik

Ei m)2 h)1

(lEi m)2 s)1) r2

Iave

Ei m)2 h)1

(lEi m)2 s)1)

Light response of primary productivity

8/20 0.42 21 0.72 (200) 0.24 (67)

Date (1999) Substrate Vmax l mol N L)1 h)1 Km lM r2

Michaelis–Menten parameters

8/19 NH4 3.00 1.68 0.68

8/19 NO3 0.65 0.50 1.00



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 ammo-

nium uptake velocities well below those at substrate

saturation (Vmax). The half saturation constant (Km) of

NHþ4 uptake was similar to NHþ

4 concentrations in

summer, whereas the value for nitrate uptake,

0.50 lMM, 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 substan-

tially 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

dividing the measured NHþ4 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 NHþ4 or

NO)3 uptake rates in the lake (Fig. 4). One-way

ANOVAANOVA 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 NHþ4 , and 0.35–

0.44 lmol N L)1 h)1 for NO)3) were not significantly

different from one another (P ¼ 0.05).

Table 3 Average rates of NH+4 and NO)

3

uptake [as estimated from direct tracer

(DT) and Michaelis–Menton (MM) tech-

niques] 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)

Date (1999)

6/3 6/29 7/25 8/20 9/18 10/19

NH+4 uptake

DT, lmol L)1 h)1 12.3 2.6 40.2 2.7 4.3 3.4

DT, lmol lg chl a)1 h)1 2.01 0.11 0.62 0.08 0.09 0.43

MM, lmol L)1 h)1 0.4 1.87 2.48 0.89 3.94 0.65

MM, lmol lg chl a)1 h)1 0.07 0.08 0.04 0.03 0.08 0.08

NO)3 uptake

DT, lmol L)1 h)1 0.34 0.05 3.26 0.4 0.04 0.01

DT, lmol lg chl a)1 h)1 0.06 0.00 0.05 0.01 0.00 0.00

MM, lmol L)1 h)1 0.08 0.09 0.09 0.01 0.7 0.14

MM, lmol lg chl a)1 h)1 0.013 0.004 0.001 0.000 0.014 0.018

N2 fixation

nmol L)1 h)1 – 0.8 9.62 1.28 0 0.38

nmol lg chl a)1 h)1 – 0.025 0.106 0.026 0 0.01

nmol (million hetero cysts))1 h)1 – 10.3 52 106.6 0 13.2

Total N incorporation

kmol (N epilimnion))1 5 (d))1 21.7 88.7 118.2 40.9 209.5 35.8

Turnover times

NH+4, h 13.8 17.9 0.1 0.8 5.9 47.2

NO)3, h 15.4 1.6 0.4 1.0 2.3 51.3

Particulate N, h 83.2 27.8 40.1 95.9 21.9 73.7

Fig. 4 Direct tracer (DT) ammonium and nitrate uptake rates at

different light intensities.



Fractional contributions estimated from in-situ

incubations

Comparison of the 5-day estimates of NO3), NHþ

4 and

N2 use over the depth of the epilimnion (Table 1)

indicated consistent reliance on NHþ4 ; 82–98% of the

inorganic N used was NHþ4 . Nitrate uptake provided

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 incor-

poration 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 phytoplank-

ton 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 consis-

tently lower than NHþ4 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 differ-

ent 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 NHþ4 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& follow-

ing a storm in September). The standard deviation of

duplicates was low, 0.1& on average. Phytoplankton

d15N was much lighter than NHþ4 and NO)

3d15N

during spring, autumn and winter. However, in

summer the phytoplankton and NHþ4 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 lMM). On one

of these, we estimated a 91% contribution to phyto-

plankton 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 NHþ4 . In

fact, phytoplankton signatures were slightly more

positive than those of NHþ4 , so that the model predic-

tions 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 NHþ4 diffusing from sediments.

Correlations

Pearson product-moment correlation analysis indica-

ted that algal biomass played a major role in deter-

mining the physicochemical characteristics of

Shelburne Pond. Secchi depth, DIC, DIN (both NHþ4

and NO)3) and molar TN : TP and DIN : TP ratios all

fell with rising chlorophyll a concentration, while pH

Fig. 5 The d15N signatures of phytoplankton, NHþ4 , and NO�

3 in

Shelburne Pond, VT, between May 1999 and March 2000. The

theoretical signature of cyanobacteria using only N2 ()2&) also

is given.



increased (Table 4). Statistically significant correla-

tions of chlorophyll a with TP and TN suggested that

both nutrients regulated algal biomass, while a neg-

ative correlation with TN : TP and DIN : TP ratios

indicated more pressure on N supplies. The relative

contribution of cyanobacteria to phytoplankton bio-

mass did not correlate with any measured environ-

mental variable, probably because the group

dominated throughout the ice-free season (and over

all conditions). Separate analysis of the relative con-

tribution of nitrogen fixers also showed no correla-

tions. 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 NHþ4 decreased. Negative

correlations between phytoplankton d15N signatures

and chlorophyll a, heterocyst abundance and cyano-

bacterial 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 atmo-

spheric nitrogen to phytoplankton nutrition in bloom-

prone 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

Table 4 Relationships between phyto-

plankton and environmental variables

found to be significant at P < 0.05 in

Pearson Product moment correlation

analysis

Dependent variable

Independent variables

Positively correlated Negatively correlated

Chlorophyll a TP*, TN*, temperature*, pH TN : TP*, DIN : TP*,

DIN*, Secchi depth, DIC

% Cyanobacteria NC NC

% N2 fixers NC NC

Cyanobacterial biomass Chlorophyll a*, TP*, TN,

pH, temperature

NO�3 , NH�

4 , TN : TP, DIN :

TP Secchi depth, DIC

N2 fixer biomass Chlorophyll a*, TP*, TN NO�3 , TN : TP, DIN : TP, DIC

N2 fixation rate Chlorophyll a, heterocysts NC

N source: % NO�3 TN : TP

% NHþ4 TN : TP

Phytoplankton d15N TN : TP

*Significance at P < 0.05.

NC, no correlations.



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, mi-

crobes 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 al-

gae, 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); NHþ4 and NO�

3 uptake rates

were well below Vmax; and negligible isotopic fraction-

ation 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 nonheterocys-

tous species such as Microcystis, Oscillatoria and Plank-

tothrix 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 cyn-

aobacteria 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

day photosynthesizing near the lake surface. There are

no data on NHþ4 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 cyanobac-

teria 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 NHþ4 over N2

(Raven, Evert & Eichhorn, 1992). We recommend that

the low DIN hypothesis be modified to allow both

nonheterocystous and heterocystous vacuolated cya-

nobacteria 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 dom-

inance 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 lMM;

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 lMM. The observed

succession pattern also was consistent with McQueen

& Lean’s (1987) earlier prediction of eukaryote dom-

inance at NO�3 : TP ratios >11 : 1 and temperatures

<21 �C. Caution must be exercised in interpreting

empirical relationships, however. Many physicochem-

ical attributes of Shelburne Pond change with season,

making indirect and spurious relationships a strong

possibility. The role of nitrate in prompting succes-

sions 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



was limiting in Shelburne Pond during 1999. Because

of dolomite in its catchment, the lake has exception-

ally 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 concentra-

tion.

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; Presing et al., 1999). In

addition, cyanobacteria appear to have lower Ik values

for photosynthesis than eukaryotes (Presing 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 meas-

urements of nitrogen fixation and DIN uptake in lakes

(Billaud, 1968; Takahashi & Saijo, 1988; Gu &

Alexander, 1993b). Comparisons of NHþ4 and NO)

3

uptake rates have been more common, and consis-

tently have shown that NHþ4 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; Presing 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 sum-

mer contributions of <10% (Billaud, 1968; Takahashi

& Saijo, 1988; Presing 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 NHþ4 , 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 hetero-

cystous cyanobacteria as well as non-fixers (Levine &

Lewis, 1987; Mugidde et al., 2004). The present study

indicated that low light also might prevent compen-

sation 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



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

MM estimates of NO�3 and NHþ

4 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 consis-

tently shallow enough (1 m) to keep phytoplankton in

a lighted region) resulted in a bias toward overesti-

mation 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 epilim-

nion 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 phy-

toplankton 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 NHþ4 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 indi-

cated substantial isotopic fractionation during extrac-

tion and concentration, which we corrected for in our

modelling. This fractionation might be related to

contamination with atmospheric ammonia or micro-

bial 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

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 cyanobacte-

rial blooms requires understanding of the circum-

stances 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 migra-

tion 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 Con-

sortium. 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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(Manuscript accepted 3 March 2004)



Do cyanobacteria dominate in eutrophic lakes because they fix atmospheric nitrogen?

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