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Joe Needoba

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Joseph Needoba | MSc Candidate



Nitrogen Isotope Fractionation in Phytoplankton: Variations in Light and Temperature Conditions


Introduction

The fractionation associated with the uptake and assimilation of nitrate by phytoplankton is a key factor in determining the d15N of organic matter. Knowledge of this process enables the application of d 15N for various studies in the marine environment. Research using d 15N in oceanographic studies include: food web analysis (Minagawa and Wada, 1987), biogeochemical processing of organic matter (Altabet and Francois, 1994), tracing anthropogenic wastewater (McClelland et al. 1997), and as a measure of past changes in surface ocean nutrient utilization (Altabet and Francois, 1994). Despite the interest in the use of stable isotopes, our understanding of the process of fractionation during phytoplankton nitrogen assimilation is incomplete.

Several studies have attempted to measure the magnitude of N isotope fractionation in phytoplankton. The magnitude of fractionation (a) for the uptake of NO3-, NO2-, and NH4+ has been measured in natural assemblages of estuarine (Montoya et al., 1991), coastal (Goering et al., 1990), and oceanic (Altabet and McCarthy, 1985) phytoplankton. These studies have reported a wide range in a for each of the forms of inorganic nitrogen. Laboratory studies have attempted to refine these measurements by measuring fractionation for isolated species under controlled conditions. This has also produced a range of values, particularly when comparing between studies. Part of the reason for this plasticity appears to be due to variations in culture conditions (Goericke et al, 1993). Variations in the degree of aeration and stirring of cultures produced large differences in the measured value of fractionation for nitrate uptake by Pheodactylum tricornutum (Wada and Hattori, 1978; Wada, 1980). Changes in a corresponding to changes in growth rate have been reported (Wada, 1980), although more recent reports claim there is very little change with respect to phytoplankton growth rate (Montoya and McCarthy, 1995). Unfortunately, these results are not directly comparable because the researchers used considerable different growth techniques.

In order to increase our knowledge of fractionation, it is clear that laboratory experiments should be conducted using similar culture conditions and techniques. There is a growing body of research using batch culture methods (Waser et al., in press) that allows for a more direct comparison of fractionation values for different forms of nitrogen, differences between species, and differences between growth conditions. Values of e for NO3-, NO2-, and NH4+ are considerably different (Waser et al., in press) and indicate that cellular differences in nitrogen uptake and assimilation have a significant influence on fractionation. Also, there are differences between species of phytoplankton grown on nitrate, and no clear trends are apparent between Bacillariophyceae, Dinophyceae, Prymnesiophyceae, or Chlorophyceae.

The physiological process that produces fractionation during NO3- uptake in phytoplankton is not known. A discrimination against 15N may occur during the transport of NO3- into the cell. Additionally, the process of assimilation of NO3- to organic nitrogen may fractionate the two isotopes, and a release of inorganic nitrogen with a high 15N/14N out of the cell would also produce a measurable fractionation. Although the two processes may occur together, the importance of either one is not known.


Proposed Research

The purpose of the proposed research is to measure the fractionation associated with phytoplankton growing on NO3- during ecologically relevant growth conditions. Under low light and low temperature conditions phytoplankton cells grow more slowly. Variation in fractionation associated with changes in these conditions has been reported (Wada, 1980), although is not confirmed (Montoya and McCarthy, 1995).

A cell's ability to store internal nitrogen varies among phytoplankton taxa, and varies depending on growth conditions (Dortch et al. 1984). This may impact the amount of fractionation, since the d 15N of the internal pool could increase, and fractionation will be expressed if nitrogen is released before it becomes assimilated. Therefore, the size of the internal pool and the amount of nitrogen released will directly affect the magnitude of fractionation. This is the first attempt to measure isotope fractionation and internal nitrate pools for phytoplankton species growing at different degrees of temperature or light limitation. Species of phytoplankton that differ in their ability to store nitrate (based on cell size and vacuole size) are chosen to provide a range in internal pool sizes. Additionally, the cultures will be monitored for release of nitrite, ammonium, and dissolved organic nitrogen (DON) in order to determine if nitrogen is being released from the cell in another form than nitrate. An isotope tracer study, using 13N nitrate, is also proposed. This will help determine the amount of NO3- that is released from the cell, rather than being incorporated after uptake.


Conclusion

The proposed study intends to determine the magnitude of nitrogen isotope fractionation for 3 species of phytoplankton grown on NO3- in ecologically relevant growth conditions. The effects of species type, growth rate, and internal nitrogen pools, will be investigated. The results are intended to identify the important physiological mechanisms responsible for nitrogen isotope fractionation during growth on nitrate.


References

Altabet, M.A., and R. Francois. 1994. The use of nitrogen isotopic ratio for reconstruction of past changes in surface ocean nutrient utilization. p. 281-305. In R. Zahn et al. [eds.], NATO ASI Series, Vol. I 17. Carbon Cycling in the Glacial Ocean: Constraints on the Ocean's Role in Global Change.

Dortch, Q., J.R. Clayton.Jr., S.S. Thoresen, and S.I. Ahmed. (1984) Species differences in accumulation of nitrogen pools in phytoplankton. Marine Biology Vol. 81 pp. 237-250

Goericke, G., J.P. Montoya, and B. Fry. 1994. Physiology of isotopic fractionation in algae and cyanobacteria. p.187-221. In K. Lajtha and R.H. Michener [eds], Stable Isotopes in Ecology and Environmental Science. Blackwell Scientific Publications.

Goering, J., V. Alexander and N. Haubenstock (1990) Seasonal variability of stable carbon and nitrogen isotope ratios of organisms in a North Pacific Bay. Estuarine Coastal and Shelf Science Vol. 30 pp. 239-260.

Mariotti, A., J.C. Germon, P. Hubert, P. Kaiser, R. Letolle, A. Tardieux, and P. Tardieux. 1981. Experimental determination of nitrogen kinetic isotope fractionation: some principles; illustration for the denitrification and nitrification processes. Plant Soil 62: 413-430.

Minagawa, M., and E. Wada. 1984. Stepwise enrichment of 15N along food chains: Further evidence and the relation between d 15N and animal age. Geochemica et Cosmochimica Acta 48: 1135-1140.

Montoya, J.P., and J.J. McCarthy. 1995. Isotopic fractionation during nitrate uptake by phytoplankton grown in continuous culture. Journal of Plankton Research 17: 439-464.

Montoya, J.P., S.G. Horrigan, and J.J.McCarthy (1991) Rapid, storm-induced changes in the natural abundance of 15N in a planktonic ecosystem. Geochim. Cosmochim. Acta Vol. 55 pp. 3627-3638

Wada, E., and A. Hattori. 1978. Nitrogen isotope effects in the assimilation of inorganic nitrogenous compounds by marine diatoms. Geomicrobiology Journal 1: 85-101.

Waser, N.D., D.H. Turpin, P.J. Harrison, B. Nielson, and S.E. Calvert. (in press). Nitrogen isotope fractionation during the uptake and assimilation of nitrate, nitrite, ammonium and urea by a marine diatom. Limnology and Oceanography (In press).


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