LIGHT ADAPTATION BY NATURAL PHYTOPLANKTON POPULATIONS, AND ITS CONTROL BY IRON LIMITATION

M.A. van Leeuwe, T. van Oijen (RUG) Introduction

The Southern Ocean is a turbulent area, with deep wind mixed layers. The Open ocean area is furthermore characterized by low concentrations of trace metals. The low biomass of phytoplankton, generally encountered in these regions, is hypothesized to be under control of the variable light climate and low iron concentrations (Chisholm et al. 1991). Iron is essential in photosynthesis, as it forms a structural component of the photosystems. An interaction of iron and light limitation can be envisioned, if iron-deplete cells are unable to use the available light. Previous experiments have shown that photosysthesis is indeed limited by iron limitation (van Leeuwe et al.1998). It was also found, however, that the pigment composition was not affected by iron limitation. This was confirmed in lab experiments, where it was found that the light utilization efficiency was target of iron limitation, whereas the absorption capacity of the cells was not affected (van Leeuwe & de Baar subm.), This led to the hypothesis that whereas on the long term photoadaptation by phytoplankton is not limited by iron limitation, short term adaptation may well be inhibited. Two aspects of photoadaptation were studied On this cruise.

Photoadaptation on a short time scale requires flexibility of the photosynthetic membranes (Kroon et al. 1994), and involves changes in cell metabolism. The integrity of the membranes is affected by iron limitation. Therefore, a quick re- sponse by the algae to changes in light conditions may be inhibited. To study the response in photosynthesis, fluorescence was measured. In addition, chlorophyll was analyzed. During periods of reduced photosynthetic activity, cells may use a storage pool of polysaccharides, to support a basic metabolism. Iron limitation results in a reduction in this pool, and thus affects the vitality of the cell under light Stress. Various cell metabolites, including carbohydrates, are studied to gain insight in the way cell metabolism is affected by iron limitation.

Material & Methods 1. Deck incubations

Deck incubations were performed to study the photosynthetic response of algae under natural conditions. At dawn, surface water was sampled using buckets, at several sites in the Polar Frontal region (Table 1). The plankton was incubated in 20L polycarbonate bottles (Nalgene) in a deck incubator, at 4 different light

intensities (100, 60, 40, 20% of incedent irradiance). After 5 hrs of daylight samples were taken for fluorescence measurements and pigment composition. In addition, cellular fluorescence and cell size were recorded using flow cytometry. At sunset, and after 5 hours of darkness, samples were taken for carbohydrates, POC/PON, chlorophyll and flow cytonietry.

2. Iron-enrichment experiments.

a. Short term incubations

Sea water was enriched with iron, to study physiological responses of the phytoplankton community. Samples were taken with clean Go-Flo's attached to a teflon coated rosette frame (Table 1). Upon retrieval on deck, the Go-Flo's were mounted to the wall of a clean cooled container. The Go-Flo's were sampled through teflon tubing, which was led through the container wall. The clean sea water was incubated in acid-cleaned 20L polycarbonate bottles (Nalgene), at 80-100 pmol photons m" s" at a 12:12h light dark regime, at 2OC. Samples were taken in the late evening, early morning. After 24 and 36 hours, samples were taken for POC, chla, cell metabolites, and samples were taken for subexperiments.

The sarnples were spiked with ^C, and placed at 20 pmol and 100 pmol photons m'* s". At regular time intewals, samples were filtered. fractionation analysis at home will be performed to record the development of the storage pool of polysaccharides over the day. In addition, at t=24h and t=36 hours, samples were taken for POC/PON, ch1.a and carbohydrates.

Samples were placed at 20 and 100 p o l photons m" s". After 5 hours, fluorescence was recorded with a dual amplitude modulated fluorometer (PSI).

After 36 hours samples were taken for ch1.a. In addition, at t=24h and t=36 hours, samples were taken for POC/PON, ch1.a and pigment composition.

b. Long term incubations.

A remaining questions is, whether or not iron limitation affects the C-N ratio of the cells. Light conditions may hereby play a crucial role (Stefels & van Leeuwe 1998).

Sea water was sampled with the Go-Flo's either mounted on the rosette frame, or attached to a kevlar wire, and by sampling from the iron fish (see de Jong et al).

The plankton was incubated in the clean cool container, in 2.5L polycarbonate bottles (Nalgene), at three different The plankton was incubated in the clean cool container, in 2.5L polycarbonate bottles (Naigene), at three different light intensities (ca. 100, 50 and 25 pmol photons m"2 s"). Growth and nutrient consumption was followed over several days (6-8). The time was given to allow any possible changes in cell metabolism, related to iron limitation, to become expressed in carbon and nitrogen content. At the end of the experiment, samples were taken for POC, PON and ch1.a.

Preliminary results.

1. Deck incubations.

The most prominent feature in the deck incubations, is the increase in cell size with increasing light intensities (Fig. 14.1). The changes in cellular fluorescence are less pronounced. A slight decrease in fluorescence with decreasing light intensities seems apparent On sunny days, On cloudy days cellular fluorescence seems to increase. The flow cytometer data have to be treated with care, especially since the delay between sampling time and measurement varied between samples, which did influence the considered Parameters. It can be concluded however, that cells do respond to changes in irradiance by varying their absorption capacity, on a relative short time scale. Changes in chlorophyll content have yet to be analyzed.

2. Iron-enrichment exweriments.

Data on the short term experiments are too preliminar to make any statements about. Considering the development of cell numbers on a short time scale, growth rates of the phytoplankton population were too low to distinguish any effect of iron iimitation (Fig. 14.2). Over a longer time Span, it is clear that growth is most optimal at the 50% light intensity, meaning ca. 40-50 pmol photons m" s". Notably, the non-enriched cells showed a faster increase in cell number than the iron-enriched cells (Fig. 14.2). The fluorescence pattems were similar for both the non-enriched and the iron-enriched cells (Fig. 14.3). Cellular fluoresence increases with

decreasing light intensity. The cell size appeared smaller for iron-deplete than for iron-replete cells. Nutrient consumption shows a different Pattern (Fig. 14.4).

Especially the phosphate data indicate that growth in the iron-enriched bottles picks up only after 4 days. At day 6, the iron-enriched cells clearly consumed more nutrients than the non-enriched cells.

It can be hypothesised that, when provided iron, the cells need some days to adjust to the new conditions, and to consume the iron. In the meantime growth appears arrested. Only when saturated with iron, growth starts again. Additional data On carbon and iron uptake might shed more led light On the processes controlling growth of the phytoplankton population at the Polar Front.

This work was supported by

J. de Jong (analyses of iron concentrations) V. Schoemann (isotope experiments)

G. Kattner, C. Hartmann, A. Ratje (nutrient analyses) M. Veldhuis & F. Brocken (flow cytometry)

K. Timmermans & B. vd. Wagt (general assistance).

Table 1. Overview of the experiments performed during ANT XVIl3

experiment date location station comments sample

1. Deck incubations

3, long term iron enrichments

3 29 March

Fig. 14.1. Cell size and cellular fluorescence as determined by flow cytometry for experiment 4. Samples were measured at t=0, t=6.5 and t=12 hours. Large cells are excluded by the flow cytometer.

0 1 2 3 4 5 6 7

--- - -- - -- - time (days) -

Fig. 14.2. Cell growth of natural phytoplankton populations over the Course of six days, for iron enriched (open symbols) and non enriched bottles (filled symbols) of experiment 3. Plankton populations were incubated at three different light intensities.

I00 50 25 100 50 % 100 50 25 100 5.0 25 100 50 25 100 50 25 control 2 nmol Fe control 2 nmol Fe controi 2 nmol Fe

4

% irradiance

-- - - - - -- - - - - -- -- - -- -- -- -

Fig. 14.3. Cell size and cellular fluorescence as determined by flow cytometry for experiment 3. Samples were measured at t=2, 4 and 6 days. Plankton populations were incubated at three different light intensities.

0 2 4 6

Fig. 14.4. Concentrations of nitrate, phosphate and silicate in the incubation bottles of experiment 3, for iron-enriched (open symbols) and non-enriched bottles (filled symbols).

Table 2: Samples for pigment distribution and for particulate carbohydrate were taken at the following stations:

In addition, several surface waters samples for pigment distribution were taken from the ship's sea water supply, covering the box (04.1-04.10), and on transect towards the continent. After filtration over GFIF Whatmann filters, samples were quick-frozen using liquid nitrogen, and thereupon stored at -80°C At the home laboratory, the samples will be analyzed using HPLC, according to Gieskes &

Kraay (1 992).

For particulate carbohydrate, several surface waters samples were taken from the ship's sea water supply, covering the box (04.1 -04.10).

Samples were filtered over GFIF filters Whatmann and stored at -20 OC. At the home laboratoty, the samples will be analyzed using GC.

References

Chisholm, S.W. & Morel, F.M.M. (1991) Limnol. & Oceanogr. 36: 1851-1864 Kraay et al. 1992 J. Phycol.28:708-712

Kroon, B.M.A. ( 1 994) J. Phycol. 30: 841 -852

Stefels, J. & van Leeuwe, M.A. (1998) J. Phycol. 34: 486-495

van Leeuwe, M.A. Timmermans, Witte, H.J., Kraay, G.W., Veldhuis, M.J.W. & H.J.W.

de Baar 1998 MEPS 166:43-52

van Leeuwe, M.A. & H.J.W. de Baar subm. Eur. J. Phycol.