Nitrogen Isotope Fractionation in Phytoplankton:
Variations in Light and Temperature Conditions
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
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
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
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.
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.
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.
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
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:
Montoya, J.P., and J.J. McCarthy. 1995. Isotopic
fractionation during nitrate uptake by phytoplankton grown
in continuous culture. Journal of Plankton Research 17:
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).