47 Introduction Blooms of the toxic dinoflagellate, Ka renia 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 f or the daily growth needs of a moderate population (3 × 10 5 cells/L) of K. brevis (Vargo et al., this Proceedings). Howeve r, it has been observed that large K. brevis b looms frequently co-occur or occur subsequent to blooms of the N 2 -fixing cyanobacteria T richodesmium sp. (Walsh and Steidinger, 2001), and correlations between the timing and magnitude of blooms of T richodesmium sp. and K. brevis in the Gulf of Mexico and the coastal Atlantic have beenmade based on historical red tide monitoring data, unpublished research results and anecdotal information (Lenes et al., 2001). T richodesmium sp. fix dinitrogen (N 2 ) 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 T richodesmium release fixed N as dissolved organic N (DON) (Glibert and Bronk, 1994), amino acids (Capone et al., 1994) and ammonium (NH 4 + ) (Mulholland and Capone, 2001; Mulholland et al., unpublished results) into the dissolved pool where it is

av ailable for uptake by other organisms. Accumulation of NH 4 + and/or DON has been observed in and around Trichodesmium b looms in the Pacific (Karl et al., 1992, 1997; Devassy, 1987), the Gulf of Mexico (Lenes et al., 2001, Heil et al., unpublished data), along the coast of Australia (O’Neil et al., submitted) and in aging cultures (Mulhollandand Capone, 2001). The objective of this study was to determine whether release of dissolved N from N 2 fixation is sufficient to fuel observed red tide blooms in the Gulf of Mexico. To do this we first needed to quantify rates of N 2 fixation by T richodesmium in the Gulf of Mexico and then determine the ecological fate of the recently fixed N 2 .

Methods To achieve our objectives, we measured the following: (1) N 2 fixation by T richodesmium

, (2) the production of dissolved N by natural populations of T richodesmium

, (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 W alton Smith in July 2001 and

a board the R/V Suncoaster in October 2001 (Fig. 1). On the Ju ly cruise, a drogue was deployed and used to follow a sur

### Does Nitrogen Regeneration from the N 2 

### Fixing Cyanobacteria T richodesmium spp. Fuel Ka r

enia brevis Blooms in the Gulf of Mexico?
Margaret R. Mulholland 1
, Cynthia A. Heil 2 , De borah A. Bronk 3 , Judith M. O’Neil 4
, and Peter Bernhardt 1

1 Ocean, Earth and Atmospheric Sciences, Old Dominion University, Norfolk, Virginia 23529-0276, USA; 2 College of Marine Science, University of South Florida, St. Petersburg, Florida 33701, USA; 3 Vir ginia 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

A bstract T

richodesmium alleviate nitrogen (N) limitation where they occur by using atmospheric dinitrogen (N 2 ). Much of the recently fixed N 2 is regenerated as ammonium (NH 4 +) and dissolved organic N (DON). This regenerated N is then available to support the growth of other cells. We hypothesized that N regenerated from N 2 fixation provides the N necessary to support blooms of Ka renia brevis in the Gulf of Mexico, and have conducted a combination of field and laboratory inv estigations to demonstrate a viable nutritional link and to quantitatively assess the role of T richodesmium in providing N to support the growth of K. brevis. Results demonstrate that T richodesmium fix N 2 at high rates with more than 50% of this new N released as NH 4 + 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 T richodesmium and other primary producers. In the field, stable isotopes were used to trace the uptake of 15 N 2 , its regeneration into dissolved N and its subsequent uptake into plankton biomass. Dialysis bags containing T richodesmium we re suspended in gas-tight incubation bottles containing K. brevis and 15 N 2 enriched water. We observed that regenerated 15 N label (as NH 4 +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 T richodesmium 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. Fi gure 1 The Gulf of Mexico study area off the West Florida Shelf. DOTGOM #1 Cruise, R/V W alton Smith, Ju ly 7–13, 2001.48 face population of T richodesmium 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 thentransferred to experimental incubation bottles. We measured gross N 2 fixation by colonies of T richodesmium usingthe acetylene reduction technique (Capone, 1993). In addition, highly enriched 15 N 2 tracer was used to follow the transfer of N among cellular (particulate) and dissolved pools (after isolating dissolved NH 4 + and DON) (Fig. 2). In a separate set of experiments, we contained Tric hodesmium 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 T richodesmium . W e added 15 N 2 and after 2 hours, measured uptake into T richodesmium and into the surr ounding plankton. We also incubated controls where we added 15 N 2 to natural populations without T richodesmium

containing dialysis bags. During one of these experiments,

we added copepods that are known to graze on Tric hodesmium to the dialysis bags to see if this would increasethe 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 N 2 fixation ranged from 0.62 to 1.02 nmol N col –1 h –1 ov er the 5-day drogue study using the acetylene reduction method (Fig. 3). However, when we measured 15 N 2 uptake, net rates of N incorporation were much lower,

r anging from 0.16 to 0.56 nmol N col –1 h –1
, than gross N 2 fixation measured using acetylene reduction. Production

rates of 15 NH 4 + (0.3–1.2 pmol col –1 h –1 ) and DO 15 N compounds from 15 N 2 we re also low, and we believe this is because our incubation times were short (2 hours) and Tric hodesmium 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

15 N 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 N 2 fixation estimated from acetylene reduction and net

15 N 2 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 N 2 fixation. To put this into the context of K. brevis’ N demand, Trichodesmium ab undance 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 . W hile background concentrations of T richodesmium are about 0.75 col L –1
, summer Tric hodesmium 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 rangeof 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 f or a moderate (3 × 10 5 cells 10 –1 ) K. brevis population undergoing 0.2 divisions d –1 . A gain, if we assume 10 hours of N 2 fixation per day and that T richodesmium b looms 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 T richodesmium densities observed during the summer and during blooms, T richodesmium can produce ample dissolved N to fuel K. brevis population growth. F

or 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 T richodesmium w as added. The appearance of label in the surrounding plankton was due to NH 4 + 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 2to 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 N 2 fixation measured using acetylene reductions (squares) and net 15 N 2 uptake (diamonds) during a cruise aboard the R/V W alton Smith in July 2001.49 w eight DON. Up to 11% of the recently fixed N 2 w as reincorporated by cells in whole water. Unexpectedly, uptakeof recently released nitrogen did not increase when copepods ( Miracia efferata ) known to feed on T richodesmium

we re added to the dialysis bags. We consider these reincorporation rates to be underestimates because we consider the production of dissolved N from 15 N 2 to be underestimated due to the problem of large intracellular metabolite pools (see above). We conclude that recently fixed N 2 from T richodesmium is released as dissolved N and can be taken up by natural populations of plankton. N 2 fixation and subsequent DON and NH 4 + r elease 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 N 2 fixation by T richodesmium and the harmful bloom-former K. brevis.

Acknowledgements Thanks to the captain and crew of the R/V W alton 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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