47

Introduction

Blooms of the toxic dinoflagellate, Karenia brevis, occur in

the oligotrophic waters of the eastern Gulf of Mexico,

where known nitrogen (N) sources are insufficient to support observed biomass accumulations (Steidinger et al.,

1998, Walsh and Steidinger, 2001). For example, estuarine

transport could supply just 5% to 20% of the N required

for the daily growth needs of a moderate population (3 ×

105 cells/L) of K. brevis(Vargo et al., this Proceedings). However, it has been observed that large K. brevis blooms

frequently co-occur or occur subsequent to blooms of the

N2-fixing cyanobacteria Trichodesmium sp. (Walsh and

Steidinger, 2001), and correlations between the timing and

magnitude of blooms of Trichodesmium sp. and K. brevis

in the Gulf of Mexico and the coastal Atlantic have been

made based on historical red tide monitoring data, unpublished research results and anecdotal information (Lenes

et al., 2001). Trichodesmium sp. fix dinitrogen (N2) at high

rates and thereby bring new N and contribute to new production in otherwise nutrient depleted waters (Capone et

al., 1997).

It has also been observed that Trichodesmium release fixed

N as dissolved organic N (DON) (Glibert and Bronk,

1994), amino acids (Capone et al., 1994) and ammonium

(NH4

+

) (Mulholland and Capone, 2001; Mulholland et al.,

unpublished results) into the dissolved pool where it is

available for uptake by other organisms. Accumulation of

NH4

+ and/or DON has been observed in and around Trichodesmium blooms in the Pacific (Karl et al., 1992, 1997;

Devassy, 1987), the Gulf of Mexico (Lenes et al., 2001, Heil

et al., unpublished data), along the coast of Australia

(O’Neil et al.,submitted) and in aging cultures (Mulholland

and Capone, 2001). The objective of this study was to determine whether release of dissolved N from N2 fixation is

sufficient to fuel observed red tide blooms in the Gulf of

Mexico. To do this we first needed to quantify rates of N2

fixation by Trichodesmium in the Gulf of Mexico and then

determine the ecological fate of the recently fixed N2.

Methods

To achieve our objectives, we measured the following: (1)

N2 fixation by Trichodesmium, (2) the production of dissolved N by natural populations of Trichodesmium, (3) N

uptake by K. brevis, and (4) the transfer of regenerated N

to co-occurring plankton during cruises in the Gulf of

Mexico aboard the R/V Walton Smith in July 2001 and

aboard the R/V Suncoasterin October 2001 (Fig. 1). On the

July cruise, a drogue was deployed and used to follow a surDoes Nitrogen Regeneration from the N2 Fixing Cyanobacteria Trichodesmium spp. 

Fuel Karenia brevis Blooms in the Gulf of Mexico?

Margaret R. Mulholland1, Cynthia A. Heil2, Deborah A. Bronk3, Judith M. O’Neil4, and Peter Bernhardt1

1

Ocean, Earth and Atmospheric Sciences, Old Dominion University, Norfolk, Virginia 23529-0276, USA; 2College of Marine Science,

University of South Florida, St. Petersburg, Florida 33701, USA; 3

Virginia Institute of Marine Science, College of William 

and Mary, Gloucester Point, VA 23062, USA; 4

University of Maryland Center for Environmental Science,

Horn Point Laboratory, Cambridge, Maryland 21613, USA

Abstract

Trichodesmium alleviate nitrogen (N) limitation where they occur by using atmospheric dinitrogen (N2). Much of the recently fixed N2 is regenerated as ammonium (NH4+) and dissolved organic N (DON). This regenerated N is then available

to support the growth of other cells. We hypothesized that N regenerated from N2 fixation provides the N necessary to

support blooms of Karenia brevis in the Gulf of Mexico, and have conducted a combination of field and laboratory investigations to demonstrate a viable nutritional link and to quantitatively assess the role of Trichodesmium in providing

N to support the growth of K. brevis. Results demonstrate that Trichodesmium fix N2 at high rates with more than 50%

of this new N released as NH4+ and DON and that K. brevis has a high affinity for reduced N sources. In addition to

these indirect lines of evidence, we have conducted a number of studies to establish direct links between Trichodesmium

and other primary producers. In the field, stable isotopes were used to trace the uptake of 15N2, its regeneration into dissolved N and its subsequent uptake into plankton biomass. Dialysis bags containing Trichodesmium were suspended in

gas-tight incubation bottles containing K. brevis and 15N2 enriched water. We observed that regenerated 15N label (as NH4+

and DON) passed through the dialysis bags and was taken up by phytoplankton outside of the bags. With these experiments we demonstrate that N released from Trichodesmium can support the growth of associated phytoplankton and possibly

K. brevis.

Steidinger, K. A., J. H. Landsberg, C. R. Tomas, and G. A. Vargo (Eds.). 2004. Harmful Algae 2002. Florida Fish and Wildlife

Conservation Commission, Florida Institute of Oceanography, and Intergovernmental Oceanographic Commission of UNESCO.

Figure 1 The Gulf of Mexico study area off the West Florida

Shelf. DOTGOM #1 Cruise, R/V Walton Smith, July 7–13, 2001.
48

face population of Trichodesmium sp. for 5 days. Trichodesmium colonies were collected from the upper 20 m

using a 202-μm, 1-m plankton net towed at speeds <1 knot.

Colonies were transferred to filtered (GF/F, nominal pore

size of 0.8 μm) seawater using inoculating loops and then

transferred to experimental incubation bottles. We measured gross N2 fixation by colonies of Trichodesmium using

the acetylene reduction technique (Capone, 1993). In addition, highly enriched 15N2 tracer was used to follow the

transfer of N among cellular (particulate) and dissolved

pools (after isolating dissolved NH4

+ and DON) (Fig. 2).

In a separate set of experiments, we contained Trichodesmium colonies in dialysis tubing with either 1 kDalton

or 100 kDalton size cut-offs and suspended the sealed

tubes in bottles containing natural plankton communities

without Trichodesmium. We added 15N2 and after 2 hours,

measured uptake into Trichodesmium and into the surrounding plankton. We also incubated controls where we

added 15N2 to natural populations without Trichodesmiumcontaining dialysis bags. During one of these experiments,

we added copepods that are known to graze on Trichodesmium to the dialysis bags to see if this would increase

the production of dissolved N compounds. During the October cruise, kinetic studies were done to estimate N uptake

by K. brevis (Bronk et al., this Proceedings)

Results and Discussion

Rates of N2 fixation ranged from 0.62 to 1.02 nmol N col–1

h–1 over the 5-day drogue study using the acetylene reduction method (Fig. 3). However, when we measured 15N2

uptake, net rates of N incorporation were much lower,

ranging from 0.16 to 0.56 nmol N col–1 h–1, than gross N2

fixation measured using acetylene reduction. Production

rates of 15NH4

+ (0.3–1.2 pmol col–1 h–1) and DO15N compounds from 15N2 were also low, and we believe this is

because our incubation times were short (2 hours) and Trichodesmium colonies had substantial intracellular metabolite

pools. Intercellular pools ranged from 0.58 to 3.42 nmol

NH4+ col–1 in an earlier study (Mulholland and Capone,

1999). The time required to fill these pools with newly fixed

15N would have been at least an hour, and this would have

resulted in the initial release of unlabeled compounds rather

than recently fixed 15N2. Based on the difference between rates

of N2 fixation estimated from acetylene reduction and net 15N2 uptake, we estimate N release rates ranging from 0.16

to 0.82 nmol N col–1 h–1, or 22% to 80% of gross N2 fixation.

To put this into the context of K. brevis’ N demand, Trichodesmium abundance was between 0.6–7.8 col L–1 during

our 5-day study, and this would result in a release of about

0.1 to 6.4 nmol N L–1 h–1. While background concentrations

of Trichodesmium are about 0.75 col L–1, summer Trichodesmium concentrations are generally much higher,

averaging 20 col L–1. During blooms, densities along the west

Florida shelf can reach >1000 col L–1 (Heil, unpublished

data). Based on rates of N uptake by K. brevis (Bronk et

al., this Proceedings), we calculate an N demand on the order

of 0.15 to 1.15 nmol N L–1 h–1. This is well within the range

of N release rates measured during our July study. Independently, Vargo et al., (this Proceedings) calculated a total

N demand of 0.056 to 0.267 mmol L–1 d–1 for a moderate

(3 × 105 cells 10–1) K. brevis population undergoing 0.2 divisions d–1. Again, if we assume 10 hours of N2 fixation per

day and that Trichodesmium blooms are of similar duration

and magnitude, during the July cruise the release rates of

dissolved N were 0.001 to 0.064 mmol L–1 d–1. If we invoke

the higher Trichodesmium densities observed during the

summer and during blooms, Trichodesmium can produce

ample dissolved N to fuel K. brevis population growth.

For dialysis bag experiments, uptake of released 15N-compounds that passed through the <1 kDalton bags, was

always greater than the controls to which no dialysis bag

containing Trichodesmium was added. The appearance of

label in the surrounding plankton was due to NH4

+ or

DON regeneration and subsequent re-incorporation (Fig.

4). Up to 2% of the recently fixed 15N2 was taken up by whole

water. Uptake of dissolved material <100 kDalton was 2

to 22 times greater than uptake of the <1 kDalton material indicating significant utilization of higher molecular

Figure 2 Pools of dissolved and cellular N and pathways for uptake, metabolism and release.

Figure 3 Gross N2 fixation measured using acetylene reductions

(squares) and net 15N2 uptake (diamonds) during a cruise aboard

the R/V Walton Smith in July 2001.
49

weight DON. Up to 11% of the recently fixed N2 was reincorporated by cells in whole water. Unexpectedly, uptake

of recently released nitrogen did not increase when copepods (Miracia efferata) known to feed on Trichodesmium

were added to the dialysis bags. We consider these reincorporation rates to be underestimates because we consider

the production of dissolved N from 15N2 to be underestimated due to the problem of large intracellular metabolite

pools (see above).

We conclude that recently fixed N2 from Trichodesmium

is released as dissolved N and can be taken up by natural

populations of plankton. N2 fixation and subsequent DON

and NH4

+ release is a significant source of nitrogen to the

plankton, including K. brevis, in the eastern Gulf of Mexico. This is the first step toward demonstrating a direct

link between N2 fixation by Trichodesmium and the harmful bloom-former K. brevis.

Acknowledgements

Thanks to the captain and crew of the R/V Walton Smith

and the R/V Suncoaster. Marta Sanderson, George Boneillo,

Michelle Watson, Susan Murasko, and Ian Hewson helped

in sample collection and analysis. Florida ECOHAB shared

data and ship time. This project was funded by the National

Science Foundation OCE-0095923.

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Figure 4 Percent of N2 fixed by Trichodesmium contained in

dialysis bags that was incorporated by plankton outside of the dialysis bags. The two bars are replicates. Controls had no 15N2 uptake

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