Document

Limnol. Oceanogr.. 36(8), 1991,1756-1771
0 1991, by the American
Society
of Limnology
and Oceanography,
Inc.
Minimum iron require.ments of marine plhytoplankton and the
implications for the biogeochemical control of new production
Larry E. Brand
LJniversity of Miami, RSMAS, 4600 Rickenbacker Causeway, Miami, Florida 33 149
Abstract
The Fe : PO., ratio at which nutrient limitation of final cell yield shifts from one nutrient to the
other was determined for 22 species of marine phytoplankton. Among eucaryotic phytoplankton,
coastal species have subsistence optimum Fe: P molar ratios of lo-* to 10-3.1, but most oceanic
species have ratios of < 1O--4,indicating that oceanic species have been able to adapt their biochemical composition to the low availability of Fe in the open ocean. In contrast, both coastal
and oceanic species of cyanobacteria have relatively high Fe : P molar ratio requirements, ranging
from 1O-1.4to 1O-2.7.A simple comparison of these requirement ratios with the ratios of the Fe
and PO, fluxes to the photic zone from deep water and the atmosphere indicates that new production
of cyanobacterial biomass is Fe limited, but new production of eucaryotic algal biomass is not.
Because of the large differences among species in their Fe requirements, especially between procaryotes and eucaryotes, changes in the relative inputs of Fe and PO, to the photic zone are expected
to lead to changes in the species composition of phytoplankton communities. Indeed, the ratio of
atmospheric to deep-water inputs of nutrients and the resulting Fe : P input ratios appear to influence
the relative abundance of unicellular cyanobacteria and Trichodesmium and their vertical and
biogeographic distributions. Because some phytoplanlcton species have adaptations that reduce
their dependence on combined N and Fe but not on P, it is concluded that PO, is the ultimate
limiting nutrient of new production of organic C on a geochemical and evolutionary time scale,
even though N and Fe are important growth rate-limiling nutrients on an ecological time scale.
What controls the rate of new production
of organic C in the ocean is of great interest
because of its influence on atmospheric 0,
and C02, sedimentation and organic burial,
and fisheries. One of the first to consider the
geochemical control of new production in
the ocean, Redfield (195 8) conducted a gedanken experiment in which he compared
the ratio of NO3 to PO, in deep water with
the ratio of N to P in marine biota and asked
which nutrient would be depleted first by
the biota if the deep water was upwelled into
the photic zone. At that time,, trace metals
were not generally considered as important
limiting nutrients because of the high concentrations observed in the ocean as a result
of inadequate techniques and serious contamination problems. As a result of greatly
improved techniques, we now know that
many trace metals are present in the ocean
Acknowledgments
This research has benefited greatly from discussions
with Bill Sunda and Bob Guillard. An earlier draft was
improved by comments from Nicholas Fisher, Francois Morel, Sallie Chisholm, and an anonymous reviewer.
The work was partially supported by NSF grant OCE
84-08672.
at extremely low concentrations
(Bruland
1983). We now have sufficient data to once
again conduct Redfield’s gedanken experiment, but this time to also consider Fe, Zn,
Mn, and Cu as potentially limiting nutrients.
Martin et al. (1976), Collier and Edmond
(1984), and Moore et al. (1984) used clean
techniques to measure the trace element
composition of plankton collected with nets
with mesh sizes ranging from 44 to 76 pm.
Wallace et al. (1977) measured the trace element composition of particulate matter in
the photic zone collected with 0.4~pm filters. These particulate elemental ratios along
with dissolved deep-water nutrient concentrations are shown in Table 1. Similar comparisons have been presented by Brand
(1986). The higher trace metal concentrations relative to P in the particles analyzed
by Wallace et al. (1977) probably reflect the
considerably smaller size fraction collected.
It is suspected that some of the material was
ino:rganic and that the larger particles collected with plankton nets in the other studies may more accurately reflect the composition 0.f living organisms. Estimates of
the nutrient ratios entering the photic zone
1756
Minimum
1757
iron reqvirements
Table 1. Nutrient ratios of plankton and deep water.
P
Fe
Plankton elemental composition (molar ratios normalized to P)
Martin et al. 1976, 64qm net
(station 78)
1
4.2 x 1O-3
Collier and Edmond 1984,
44-pm net
1
10-2
Moore et al. 1984, 76-pm net
1
1.2 x 10-2
Wallace et al. 1977, 0.4~pm filter
(station 5, normalized to N,
assuming N : P = 16)
5.1 x 10-2
Rough avg
(t’
10-2
Dissolved nutrients in deep water (molar concn)
Atlantic Ocean
Bruland and Franks 1983
1.2x 10-h
Symes and Kester 1985
2 x 10-g
Pacific Ocean
Bruland and Franks 1983
2.7 x 1O-6
Gordon et al. 1982
10 9
Molar ratios of dissolved nutrients in deep water (normalized to P)
Atlantic
1
1.7 x 10-3
Pacific
1
3.7x10-4
based on the deep-water nutrient concentrations (Table 1) are only rough approximations because the actual source of nutrients for new production
is from the
pycnocline just below the photic zone, but
the ratios do not differ by much and the
basic argument is not altered.
By the same logic used by Redfield (19 5 8),
a comparison of deep-water nutrient molar
ratios to plankton elemental composition
ratios leads to the conclusion that plants in
the photic zone would deplete the upwelling
water of Fe long before the other nutrients.
N03, P04, Zn, and Mn would all be depleted
at about the same time. There is more than
enough Cu. Indeed it has been argued that
upwelled Cu could be toxic to phytoplankton (Sunda et al. 198 1; Brand et al. 1986;
Coale and Bruland 1988). A simplistic conclusion to be drawn from this comparison
of plankton elemental composition
and
deep-water nutrient concentrations is that
Fe availability
controls new production of
organic C in the ocean, not PO, as concluded by Redfield (19 5 8). This conclusion
seems reasonable from an evolutionary perspective. During the first half of the history
of life when most basic biochemistry
evolved, the ocean was anoxic. As a result,
Zn and Cu were less soluble while Fe and
Mn
Zn
Cu
1.76 x lO-3
6.7 x 1O-4
7.8x
4x 10-3
9 x 10-4
3.5 x 1o-4
3.4 x 10-4
5.5 x 1o-4
1.6x 1O-4
3 x 10-J
2x 10-3
5.3 x 10-3
1.5x10-3
6 x 1O-4
2x
4x10-4
10-4
10-9
7 x 10-10
2x
10-9
8 x 1O-9
2x 10-10
4x
10-9
1.7x10-3
3 x 10-3
5.8x1o-4
7.5 x 10-S
1.7x 10-3
1.5x10-3
Mn were much more soluble in seawater
and available
to phytoplankton
(Brand
1986). It is estimated that trace metal concentrations in the anoxic ocean were around
10B3 M Fe (Osterberg 1974), 10m5 M Mn,
lo-l1 M Zn, and lo-l7 M Cu (Ochiai 1978).
Given the high concentration of Fe relative
to other trace metals, it is not surprising that
it is used more than any other trace metal
for various biochemical functions (Osterberg 1974; Egami 1975). The shift to an
aerobic ocean resulted in a drastic reduction
in Fe solubility and availability
(from 1O-3
to 1O-9 M) and quite possibly led to Fe becoming the geochemically limiting nutrient.
This gedanken experiment is incomplete,
however, because a significant fraction of
the Fe entering the photic zone of the open
ocean comes from the atmosphere, not just
from deep water (Duce 1986), which makes
the analysis much more difficult because one
must now measure and compare flux rates
from the atmosphere and the deep ocean,
and not just standing concentrations in deep
water. Table 2 shows some estimates of these
fluxes to the photic zone of the Pacific Ocean.
Duce (1986) has presented similar comparisons to demonstrate the importance of atmospheric inputs. These ratios are not only
more difficult to measure but also more
Brand
1758
Table 2. Nutrient flux rates to the Pacific photic zone (mol m-2 d-l).
=.
-.
North Pacific central gyre
Flux from deep water
(Martin and Gordon 1988)
Flux from atmosphere
(Duce 1986)
Southern Ocean
Flux from deep water
(Martin et al. 199Ob)
(P flux estimated from
N:P= 16)
Flux from atmosphere
(Martin 1990)
-
N
1’
4x10-4
3x10-5
8 x 10 -,j to 2.6 x 1O-5
6.3 x 1O-3
variable because of the large differences in
the relative input of atmospheric dust and
deep water to the photic zone in different
parts of the ocean. Because of geochemical
fractionation
between ocean basins, the
Fe : P dissolved nutrient ratio is 5 times
higher in the Atlantic than the Pacific (Table
1). At the same time, it appears that the
atmospheric Fe input is higher in the Atlantic than the Pacific because the Atlantic
basin is smaller and in closer proximity to
land. Buat-Menard and Chesselet (1979) estimated an atmospheric Fe input of 1.6 x
1O-6 M m-2/d-1 to the Sargasso Sea and
Duce ( 1986) estimated 2-8 x 1Oe7 M Fe
m-2/d-1. In contrast, Duce (1986) estimated
only 8 x lo-* to 1.6 x 1O-7 M Fe m-2/d-1
to the North Pacific central gyre. As a result,
Fe limitation is much less likely in the Atlantic and so efforts to detect Fe limitation
are focused on the Pacific. The lowest Fe : P
flux ratio to the photic zone is expected to
be in the Southern Ocean or the equatorial
Pacific where upwelling is strong but there
is very little continental dust input (Martin
and Gordon 1988; Martin 1990). There the
Fe : P molar ratio supplied to the photic zone
would be - 10m4.
A complicating factor is the wide array of
chemical forms of Fe in seawater- some
available to phytoplankton
and some not
(Rich and Morel 1990; Wells 199 1). Generally most but not all “dissolved”
Fe (that
which passes a 0.4-pm filter) is available to
phytoplankton.
Some of the inorganic colloidal Fe that passes through a 0.4-pm filter
can be in a chemical form that does not
dissolve sufficiently to supply significant
5x
IO-9
to 1.2x 10-s
(3.9 x 10-d)
-~
Fe
4.3 x IO-9
8x 1O-8to 1.6x lo-’
2.5 x 10-S
1.2x10-8
amounts of Fe to phytoplankton
(Wells
199 1). On the other hand, some forms of
particulate Fe are available to phytoplankton through dissolution and photochemical
reduction to Fe 2+ (Rich and Morel 1990;
Wells 199 1). Early work (Hodge et al. 1978)
indicated t,hat only - 1% of the Fe in atmospherically derived particles would dissolve in seawater, but more recent work suggests that 10 (Moore et al. 1984) to 50%
(Zhuang et al. 1990) can dissolve, depending
on conditions. Overall, it appears quite possible that a significant fraction of the particulate Fe in seawater is also available to
phytoplankton. Although we still have much
to learn about the chemistry of Fe in seawater, I will assume here that all upwelled
dissolved Fe but none of the upwelled particulate Fe is available to phytoplankton.
Only dissolved Fe concentrations are shown
in Table 1, which does not introduce much
error as particulate Fe concentrations in deep
water of th.e open ocean are similar to concentrations of dissolved Fe (Gordon et al.
1982; Martin and Gordon 1988) and a high
fraction of dissolved Fe in offshore waters
seems to be available to phytoplankton
(Wells 199 1).
There are also complications in estimating the nutrient needs of phytoplankton.
Careful elemental analysis of particulate
matter in the photic zone by Martin et al.
(19’76), Collier and Edmond (1984), and
Moore et al. (1984) indicated a Fe : P molar
ratio of - 100~ (see Table I). Data on the
elemental composition
of particles in the
ocean, however, do not necessarily reflect
the composition of phytoplankton
because
Minimum
iron requirements
such particles also include detritus, zooplankton,
and inorganic
matter (Banse
1974). The higher trace metal content of the
smaller particles (Table 1) analyzed by Wallace et al. (1977) probably reflects this problem. Furthermore, nutrient content does not
necessarily indicate minimum nutrient need.
The nutrient uptake data of Martin and
Gordon (1988) and Martin et al. (1990a)
indicate a Fe : P molar ratio of 4 x 1O-4 to
10-3. Such data on nutrient uptake rates
however do not necessarily indicate minimal nutritional needs or balanced elemental
ratios because of luxury uptake (Droop 1974;
Goldman and McCarthy 1978). The laboratory study by Anderson and Morel ( 1982)
with the coastal diatom Thalassiosira weissjlogii suggests a minimum Fe : P molar ratio
requirement of 8 x 10e4. The extrapolation
of the Fe requirements of this coastal diatom to phytoplankton
in general assumes
that all phytoplankton have similar requirements and that the “extended Redfield ratio” (Table 1) is rather uniform among phytoplankton species.
The uncertainty over the minimum Fe : P
molar ratio quota needed by phytoplankton
provides the rationale for this study. My
primary purpose was to determine the Fe : P
molar ratio at which biomass production
limitation shifts from Fe to P limitation in
a wide variety of phytoplankton,
particularly those that live in oceanic regions with
strong upwelling and relatively little dust
input, where Fe limitation
is most likely.
Given the potential problems with using elemental composition of particles in the field
to indicate nutritional need, the data in Table 1 cannot exclude the possibility that other trace metals such as Zn, Mn, or Cu could
limit new production in the ocean. Experiments (Brand unpubl.) have shown that the
Zn and Mn cell quota requirements relative
to P are considerably less than what is provided to the photic zone. Presented here are
the results of experiments on the Fe requirements of marine phytoplankton.
Methods
Clonal cultures of phytoplankton
of various phylogenetic origins were isolated from
various coastal and oceanic habitats or obtained from the Provasoli-Guillard
Center
for Culture of Marine Phytoplankton (Table
1759
3). Seawater for the experiments was collected from the northern Tongue of the
Ocean, Bahamas, from in front of the ship
as it moved forward at - 1 knot. The water
was collected with a pumping system that
allowed the seawater to contact only Teflon,
polyethylene, and a 30-cm section of silicone tubing through a high-speed peristaltic
pump. The seawater and individual
nutrient stock solutions were all tyndallized separately in Teflon bottles.
Nutrients added to the seawater were: 1Om3
M NaN03, either 1Ow6 or 2 x 10m6 M
NaH,PO,, lo-* M MnSO,, lOUs M ZnSO,,
1O-g M CoSO,, 1O-g M CuSO,, lo-* M
H,SeO,, 1O-* M thiamine, lo-* M biotin,
and 10Bg M vitamin B12. Concentrations of
Fe-EDTA added were: none, 1O-lo M, 1O-g
M, lo-* M, or 10e7 M. EDTA was added
to a concentration of 10m7M to ensure that
Fe and other trace metals did not precipitate
to a form unavailable to the phytoplankton,
although some Fe probably precipitated at
the highest concentration. A higher concentration of EDTA was not used to avoid the
problem of overchelation at low Fe concentrations that could prevent the phytoplankton from taking up most of the Fe. The free
ionic activities of the other trace metals in
the culture media are estimated as follows:
Zn- 10-g.5; Mn- lo-*; Cu- 10-12a7; and
Co- 1O-g.7. A comparison with the data of
Brand et al. (1983, 1986) indicates that the
cultures were not nutritionally
limited by
these metals nor affected by any trace metal
toxicity. Background concentration of PO4
in seawater from the Tongue of the Ocean
was determined with a Technicon CSM6
AutoAnalyzer.
Background [Fe] was determined by comparing the biomass yield with
no, lo-lo, and 10hg M Fe added to the seawater. The experiments were conducted in
25- x loo-mm polycarbonate test tubes with
screwcaps. The experimental test tubes were
cleaned with detergent and repeated rinses
and soaks of dilute HCl and deionized water
(18 Ma), microwaved for sterilization (Keller et al. 1988), and finally rinsed with tyndallized seawater from the Tongue of the
Ocean before experimental culture media
was added.
Experiments were conducted at 2 lo-23°C.
A light intensity of 59 PEinst m-2 s-* was
provided by “cool-white”
fluorescent bulbs
Brand
1’760
Table 3. Origins of experimental cultures.
Clone
Species name
Coccolithophores
A2362
Calcidiscus leptoporus
Al387
Emiliania huxleyi
451 B
Emiliania huxleyi
A222 1
Gephyrocapsa oceanica
cocco 2
Pleurochrysis carterae
Se62
Syracosphaera elongata
A2390
Umbilicosphaera sibogae
Dinoflagellates
Amphi
Amphidinium carterae
WT8
Gymnodinium simplex
Al569
Peridinium sp.
Exuv
Prorocentrum minimum
Al379
Thoracosphaera heimii
A2414
Gymnodinium sp.
Other eucaryotes
MC-l
Mantoniella sp.
A2167
Micromonas sp.
OLISTH
Heterosigma akashiwo
Cyanobacteria
SYN
Synechococcus bacillaris
Al601
Synechococcus sp.
A2169
Synechococcus sp.
A2357
Synechococcus sp.
A2367
Synechococcus sp.
DC2
Synechococcus sp.
Lcxation
Date
Isolator
0”24.1O’S, 113”17.84’W
23”48.8’N, 89”45.7’W
Oslo fjord, Norway
08”53.7’N, 79”3 1.7’W
Woods Hole, Massachusetts
?
24”19.51’N, 82”12.22’W
15 Jan 90
23 Apr 80
?
26 Ju183
Jul 58
?
4 Jan 90
Brand
Brand
Paasche
Brand
Pintner
Droop
Brand
Great Pond, Falmouth, Massachusetts
9”48.5’N, 89”14.5’W
23”56’N, 8234’W
Great South Bay, Long Island
23”48.9’N, 89”45.7’W
5”07.92’S, 149”09.12’W
Sep 54
Guillard
1958
28 Jan 81
Jul58
23 Apr 80
23 Jan 90
Dodson
Brand
Pintner
Brand
Brand
North Pacific central gyre
05”08.l’S, 81’43.1’W
Long Island Sound
17 Feb 73
20 Ju183
1952
Lewin
Brand
Conover
Milford, Connecticut
19”45’N, 92”:25’W
05”00.5’S, 84”29.4’W
Northern Tongue of the Ocean
24”19.51’N, 82”12.22’W
33”44.9’N, 67”29.8’W
1957
1980
21 Ju183
16 Nov 84
4 Jan 90
1978
Guillard
Brand
Brand
Brand
Brand
Brand
with 2 x lOem M PO, to test if it was the
other limiting nutrient when Fe was not limiting. The culture with no Fe added was used
as a bioassay to estimate the highest possible background concentration of Fe in the
seawater used.
Because the nutrient ratio at which Fe
limitation shifted to PO, limitation was determined in stationary phase, it is equivalent to the optimum nutrient ratio at zero
growth rate of Rhee and Gotham (1980).
COPY.
The overall design of the experiment was Nutrient cell quotas are necessarily larger at
higher growth rates. If the quantitative
reto determine final cell yield in different cullationships between cell quotas and growth
ture media with either no Fe added or consimilar for two difcentrations ranging from 1O-lo M up to 1Oe7 rates are proportionally
M. At some point, Fe concentrations were ferent nutrients, the optimum nutrient ratio
would be the same at all growth rates. Terry
no longer limiting cell yield and PO4 became
et al. (1985) and Elkfi and Turpin (1985)
the limiting nutrient of cell yield at a conhave demonstrated that the optimum N : P
centration of 10M6M. Minimum cell quota
can then be calculated from the final cell ratio varies with growth rate but the variation is not large. The optimum N : P ratio
yield and the initial total nutrient concentration, assuming the nutrient-starved
cells of Selenastrum minutum varied from 16.4
to 2 1.5 over a range of growth rates of Otake up essentially all of the nutrient. An
1.‘7 div. d-l (Elrifi and Turpin 1985). For
additional culture with 1Oe7M Fe was grown
with a 12 : 12 photoperiod.
Experimental
cultures were grown as sequential replicates
for 66 d and monitored by measuring in
vivo fluorescence with a Turner lo-OOOR
fluorometer.
Once the fluorescence measurements indicated that the cultures had
reached peak abundance, cell abundance was
determined by microscopic counts with hemacytometers or settling chambers and either direct light or epifluorescence micros-
Minimum
iron requirements
Pavlova lutheri it varied from 37.9 to 48.9
over a range of 0.55 to 1.21 div. d-l. Although both Fe (Davies 1970; Harrison and
Morel 1986; Sunda et al. 199 1) and PO,
(Rhee 1982; Terry et al. 1985; Elrifi and
Turpin 1985) cell quotas are higher at faster
growth rates, no data set at present shows
how the optimum Fe : P ratio varies with
growth rate. Depending on the relative increase in Fe and P quotas needed to sustain
higher growth rates, the optimum Fe : P ratio at higher growth rates could be higher
or lower than the subsistence optimum Fe : P
ratio at zero growth rate measured in these
experiments.
Final yields are presented here as cells per
seawater volume, which is the proper measurement for addressing questions of evolutionary adaptations (genome per nutrient)
but not necessarily for questions of C transport from the atmosphere to deep water.
With respect to new production of organic
C, there is undoubtedly a bias, but the C
per cell variation is not large enough to alter
the general conclusions presented here. C
per cell varies much less than other nutrients (Goldman and McCarthy 1978).
Results
The final cell concentrations achieved in
the different nutrient regimes are shown in
Figs. l-4. Where Fe limitation was achieved,
the sloping line on the left in the figures is
given the theoretical slope along which Fe
is the limiting nutrient, not the best fit to
the data points. Slopes vary among figures
only because of differences in the ranges of
cell concentrations on the vertical axis. The
intersection of the sloping lines on the left
in the region of Fe limitation and the horizontal lines in the region of PO4 limitation
indicates the subsistence optimum Fe : P
molar ratio at which there is a shift from
Fe to PO, limitation
of the maximum cell
yield. The vertical lines separate the data at
the higher PO, concentrations
that demonstrate that PO4 is indeed the limiting nutrient when Fe is not limiting.
Background concentration of PO4 was < 3
x 10m8M. Based on the fact that 1O-lo M
Fe additions led to small increases in cell
yield and 1O-g M Fe led to order-of-magnitude increases in those species that were
1761
Fe limited, it is concluded that background
concentrations of Fe in the seawater media
were - lo-lo M. It is suspected that Fe concentrations are less than were initially present in the water when it was collected as a
result of precipitation,
adsorption to the
polyethylene walls of the storage containers,
and uptake by bacteria growing on the walls
of the containers over the 5 yr of storage.
The Fe : P molar ratio at which Fe limitation of cell yield shifts to PO, limitation
is quite different for different species of phytoplankton. Among eucaryotic phytoplankton, the subsistence optimum Fe : P ratio
ranges from - 10-3.4 in Micromonas sp. to
< 1O-4 in most of the oceanic species (Figs.
1, 2) but from - 1O-2 to 10-3.1 in coastal
species (Fig. 3). The lowest Fe : P molar ratio attained in the experimental media because of the background concentration
of
Fe in the seawater was - 10m4.Most of the
oceanic phytoplankton species show no signs
of Fe limitation
even at this ratio. These
data indicate that many eucaryotic phytoplankton living in low Fe environments have
adapted by having a biochemical composition with very little Fe and that natural
selection has led to oceanic species needing
less Fe than coastal species. Although diatoms were not examined in this study, Sunda et al. (199 1) investigated the Fe requirements of two closely related diatoms, one
from estuarine waters and one from the open
ocean, and found essentially the same result
with very different methods.
All the procaryotic cyanobacteria tested
here, both coastal and oceanic, need relatively high Fe : P molar ratios, ranging from
lo-la4 to 10m2.’ (Fig. 4). Some habitat-related pattern can be discerned but it is weak.
The only clone (A2169) from the Pacific
Ocean, where Fe : P molar ratios in deep
water are lowest, has the lowest Fe : P molar
ratio requirement. The only clone (SYN)
from coastal waters is one of three with the
highest subsistence optimum Fe : P ratios.
Overall however, the differences among the
cyanobacteria do not relate as well to their
habitats as do those of the eucaryotic phytoplankton species. Even the cyanobacterial
clone with the lowest subsistence optimum
Fe : P ratio has a ratio higher than most eucaryotes and higher than the nutrient ratios
Brand
.-_-Calcidiscus
I
I
leptoporus
I
Fe
_______
IO-M
lo-SM
lo-%4
10.7M
lo-7M
P
lo-%i
lo-6M
l@M
lO%l
10-GM
2xlO43M
10s
107
Emiliania
Gcphyrocapsa
huxleyi (451 B)
oceanica
I 8
ij
u
‘OR
1
0
r---&l
l@
105
FO
10s
___.___
lo-‘01
lo-%I
IO-f‘M
lo-7M
104%
10-W
10-W
lo%
lo-7M
Fe
2x10-6M
P
-.-_-._
10-l’)M
W’M
IO-EM
10.‘M
lO-7M
lo-6M
lo-GM
2x10-m
1
Umbilicosphaera
sibogae
I
Fe
P
_______
IoJOM
lo-f’M
10-EM
lo-7M
10.71
lo43M
lo45M
lo4M
104M
lo-6M
2x10&M
Fig. 1. Final cell yields of oceanic coccolithophores in culture media with various concentrations of Fe and
PO, added.
Minimum
Gymnodinium
Fc
P
_._._I
1w3M
Peridinium
simplex
lO-‘M
10%
10-m
lOal
Thoracosphaera
1763
iron requirements
lo-SM
lO-7M
lo-7M
sp.
FC
_______
W’-‘M
lo.%
lo-EM
10.7M
lo-7M
P
lo-GM
1043M
lo-rrM
10-6~
104~
2~10-6~
Gymnodinium
sp.
heimii
I
I
I
’
8
I
cl
I
a
’
l@
9
e
cl
0
0
0
8
0
0
El’
o
‘I
8
/r----j
6
0
I
I
I
104
Fe
P
I____.
104M
lo-l”M
10%
lo-f’M
lo-7M
lo.7M
FC
____.__
10-M
10%
IO-EM
lo-7M
10.7M
IO-al
lo-SM
IO-GM
1043M
2X104M
P
lo-GM
lo-6M
lo-fiM
104M
lo-GM
txlO4M
Micromonas
sp.
I
I
10s
Ft!
P
_..____
104M
10.1”M
1043M
lo-f’M
lO%I
10.RM
lo-6M
lO-7M
lW3M
lo-7M
2xlO4M
FC
_..----
lO-10M
10%
IO-RM
1O’M
lo-7M
P
IO-CM
104M
lo-6M
1043M
106M
2x10%
Fig. 2. As Fig. 1, but of oceanic dinoflagellates and prasinophytes.
1764
Brand
1
Plourochrysis
carterae
Syracosphaera
elongata
1Or
104
i
10s
l
101
Fe
-.._...
10-M
IO-f’M
lo-EM
lo-7M
lO-7M
P
104M
10%
1043M
104M
lo-‘3M
2x10-EM
I------Fe
10-M
10%
lo-8M
IO-71
lo-7M
l@M
10%
10-W
10-W
2x104M
-
-
I
Prorocentrum
minimum
I
f-+-G
8
c
Fe
P
mm.m.-.
10-M
10%
W’M
lO-7M
104M
10%
1O”sM
104M
10-w
--L--
105
Heterosigma
akashiwo
8
FC
___..__
10-M
10-9M
lo-8M
lo-'&l
1’
IO-fiM
104M
10-w
lo-‘%I
10.‘%I
lo-7111
2x10-6M
Fig. 3. As Fig. 1, but of coastal coccolithophores, dinoflagellates, and a raphidophyte.
10-7M
2x10-w
Minimum
1765
iron requirements
Synechococcus sp. (A1601)
Fe
P
I
-.---mm
lO-‘M
1W8M
lo-EM
lo-‘M
lo.‘M
Fe
-_._...
10.lOM
IO-BM
104M
lo-7M
lo-7M
lo-6M
lo-6M
lo-%I
lO+‘M
lo-6M
2~10%
P
lo-GM
10%
mm
1043~
10-6~
2x104~
I
I
Synechococcus sp. (A2169)
107
B
i
107
10s
0
I
I@
I
I
10s
Fe
P
-<.mm.
10-W
lo-‘OM
llwm
10%
10%
104M
10-6~
lO-7M
10-6~
104
.
lo.7M
Fe
____._.
10-M
10%
104’M
lo-7111
lo-7M
2~10-6~
P
104M
104M
104M
lo-6M
lO-‘JM
2xlO43M
-t
1Oe
Synechococcus sp. (A23671
107
il
s
10
I
104
1Oa
Fe
_m_I---
1o”oM
10%
10.*M
107M
lo-7M
FC
_._____
lo-l”M
10%
104M
10-7M
lo-7M
P
1043M
lo.%I
104M
lo-%I
106M
2x10-Q
P
10-6M
10-W
IO-GM
10-w
104M
2x104%
Fig. 4. As Fig. 1, but of coastal and oceanic cyanobacteria.
1766
Brand
transported to most of the oceanic photic
zone. These data indicate that oceanic cyanobacteria have been unable to evolve a
significantly lower optimum ratio like the
eucaryotic phytoplankton.
Discussion
The coastal-oceanic difference in the minimum Fe requirements for biomass production of eucaryotic phytoplankton
reported here is similar to that found by Brand
et al. (1983) who observed that growth rates
of oceanic phytoplankton
are less limited
by low concentrations of Fe than those of
coastal species. Clearly, one cannot extrapolate the Fe requirements of coastal species
to oceanic species or to phytoplankton
in
general. The significant difference between
coastal and oceanic eucaryotic phytoplankton is most likely the result of natural selection, indicating that Fe is an important
environmental
factor. It appears that many
oceanic eucaryotic phytoplankton
maintain
‘high growth rates at low Fe concentrations
by having a biochemical composition with
a much lower Fe quota requirement, which
agrees with the ideas of Hudson and Morel
(1990) that Fe uptake rates may be near the
maximum
possible because of diffusion
limitation.
Although the biochemical nature of the
adaptation is not known at this time, it does
appear that oceanic eucaryotic phytoplankton have evolved very low Fe cell quota
requirements in response to the extremely
low Fe concentrations found in many parts
of the open ocean. The biochemical mechanism is of great interest because Fe is found
in such a wide array of essential molecules
such as Fe-S proteins, cytochromes, oxygenases, hydroperoxidases,
and Fe flavoproteins. In a few cases, other metals have
been shown to substitute for Fe in certain
enzymes or alternative enzymes have been
shown to need no metal ion (Raven 1988).
Fe-containing cytochrome c can be replaced
by Cu-containing plastocyanin and Fe-containing ferredoxin can be replaced by flavodoxin which contains no metal in some
species. In a similar fashion, Price and Morel (1990) have found that Cd and Co can
substitute for Zn. Studies are currently underway to determine to what extent other
metals can substitute for Fe in oceanic phytoplankton species in Fe-poor water.
Also of interest is why all cyanobacteria,
even oceanic species, need rather high
amounts of Fe in the cell. Why have oceanic
cyanobacteria been unable to evolve a low
Fe quota like the oceanic eucaryotes? As
most of the Fe is involved in redox reactions
in cytochromes or Fe-S proteins in the chloroplasts and mitochondria
(Raven 1988)
one would expect most of the Fe to reside
in these organelles rather than in the cytosol.
Indeed it is estimated that 80% of the Fe in
higher plants is in the chloroplasts (Marschner 1986). Therefore, it is hypothesized that
the Fe: P molar ratios in cyanobacteria,
chloroplasts (evolutionarily
endosymbiotic
cyanobacteria),
and mitochondria
are all
high and perhaps rather similar and that the
Fe : P ratio in the eucaryotic cytosol is much
lower, resulting in an overall lower ratio for
eucaryotes. There may be a lower limit to
the Fe : P molar ratio in procaryotes and
organelles packed with electron transport
chains, but eucaryotes can achieve considerably lower ratios by having a relatively
large amount of cytosol with little Fe. This
is <surely not the complete explanation, because eucaryotic algae such as Mantoniella
and Micromonas that are almost as small
as cyanobacteria with much of the cell volume occupied by organelles (Thomsen 1986)
are still able to grow well with very little Fe
(see Fig. 2).
The answer to Redfield’s question-which
nutrient is depleted first in the photic zone depends on the competition between procaryotes and eucaryotes and the relative
abundance of the two. It may well be that
competition
results in the relative abundance of the two groups shifting, so that the
composite Fe : P molar ratio composition of
the community reflects the input ratio. It
does appear that cyanobacteria comprise a
hi,gh fraction of the phytoplankton
community in the most stratified waters (Waterbury et al. 1986; Glover et al. 1988; Siegel
et al. 1990; Krupatkina
1990; Ondrusek et
al., 199 1) where the ratio of atmospheric to
deep-water input and the resulting Fe : P input ratio should be the highest. In contrast,
Waterbury et al. (1986) had a hard time
finding any cyanobacteria in the Southern
Minimum
iron requirements
Ocean where the ratio of atmospheric to
deep-water input and the resulting Fe : P ratio should be about the lowest anywhere in
the ocean. El-Sayed (1988) reported that picoplankton are a relatively small fraction of
the phytoplankton
community in open waters of the Southern Ocean. Similarly, procaryotic cyanobacteria are a larger fraction
of the phytoplankton community in the upper photic zone (Longhurst and Harrison
1989; Bidigare et al. 1990; Siegel et al. 1990)
where the atmospheric to deep-water input
and Fe : P ratios are relatively high, than at
the bottom of the photic zone, where the
Fe : P input ratio is expected to be lower.
Although more data are needed, these latitudinal and vertical gradients indicate that
the ratio of procaryotes to eucaryotes in
phytoplankton
communities
may be partially controlled by the Fe : P ratio of nutrient inputs to the photic zone and therefore
also ultimately
controlled by the relative
importance of atmospheric dust to deepwater input of nutrients.
It appears likely that Fe influences the
distribution
of N,-fixing cyanobacteria as
well. Fanning (1989) has examined GEOSECS and TTO surface nutrient data sets to
determine whether NO3 or PO, is most depleted to below detection limits in the photic zone. Overall, NO3 and not PO, is depleted the greatest amount at most stations
around the world, with the exception of areas downwind of North America and Asia,
where PO4 is depleted more than NOS. He
argued that most of the ocean is N limited
but is shifted to P limitation
where it receives combined N from continental sources
as a result of fossil fuel combustion.
Another explanation for this pattern is
related to the fact that the input of Fe to the
ocean in atmospheric dust would have a
distribution pattern similar to that of combined N from continental sources. A comparison of the map of NO, vs. PO4 depletion
presented by Fanning (1989) with the maps
of atmospheric dust input (a reasonable indicator of Fe input) as well as combined N
input from the atmosphere (GESAMP 1989)
reveals striking similarities. Because of the
high Fe requirement of N2 fixation (Rueter
1988; Raven 1988), it is hypothesized that
N2 fixation cannot alleviate local N limi-
1767
tation in most areas of the open ocean, but
can do so in areas receiving significant
amounts of atmospheric Fe, resulting in PO4
depletion downwind of North America and
Asia, Maps of the global distribution
of
Trichodesmium
abundance
(Carpenter
1983) show higher abundance downwind of
North America and Asia, similar to the maps
of dust input and surface PO, depletion.
While the direct input of additional combined N from the atmosphere should not
be discounted, the role of Fe in supporting
N2 fixation certainly deserves consideration. The input of both atmospheric N and
Fe can lead to PO, depletion in the photic
zone. In support of the hypothesis of Fe
limitation is the fact that additional N from
the atmosphere would deter N2 fixers such
as Trichodesmium, whereas Fe enrichment
would enhance their growth. The global distribution of Trichodesmium tends to support the idea of Fe as an important limiting
nutrient of N2 fixation on an ecological time
scale.
It is hypothesized that Fe became the limiting nutrient of new production when the
ocean shifted from being anoxic to being
well oxygenated and Fe concentrations decreased by a factor of a million. New production of cyanobacteria has been limited
by Fe ever since, but the eucaryotes, which
evolved at the time of the anoxic-oxic shift,
appear to have evolved a biochemical composition that needs much less Fe. This adaptation seems to have reduced or eliminated Fe limitation and caused a shift back
to PO4 being the nutrient that limits global
new production on a geochemical time scale.
The present data do not demonstrate with
absolute certainty that the minimum Fe requirements of oceanic eucaryotic phytoplankton are low enough to prevent Fe limitation because of uncertainties over exactly
what fraction of the Fe in the ocean is chemically available to phytoplankton
and how
much the optimum Fe : P ratio varies with
growth rate. Therefore we cannot at this time
completely rule out Fe as the ultimate limiting nutrient on a geochemical time scale.
If we assume that very low Fe quotas are
biochemically
feasible however, then natural selection would lead to optimum Fe : P
ratios that are just low enough to alleviate
.
1768
Brand
most Fe limitation, and Fe and PO, would
end up equally limiting to new production.
There is no selective pressure for a still lower Fe : P ratio quota even if it is biochemically feasible.
This prediction of Fe and P being roughly
equalling limiting of new production (along
with N because of the balance between N2
fixation and denitrification)
is similar to the
argument of Brand et al. (1983) that the
growth rates of phytoplankton
would be simultaneously limited by several nutrients
rather than just one. To the extent that a
lower Fe cell quota can evolve should a biogeochemical shift provide the selective pressure, Fe cannot be considered the ultimate
limiting nutrient on a geochemical and evolutionary time scale, despite its importance
on ecological time scales. PO, remains the
geochemically
limiting
nutrient because
phytoplankton
have not evolved a way to
reduce their dependence on this resource.
Although it appears likely that Fe does still
limit the biomass of cyanobacteria, there
are two reasons for believing that their direct contribution
to global new production
is small. They are relatively sparse compared to eucaryotes at the bottom of the
photic zone (Longhurst and Harrison 1989;
Bidigare et al. 1990; Siegel et al. 1990) where
most new production is thought to occur
(Coale and Bruland 1987; Small et al. 1987),
and their small size is thought to lead to
long food chains with most of the organic
C produced being recycled in the photic
zone, not exported to deep water (Michaels
and Silver 1988).
It is argued that neither N nor Fe can be
the ultimate geochemically limiting nutrient because some component of the phytoplankton community
has been able to
evolve a reduced dependence on them. Some
cyanobacteria can grow without any combined N by fixing dinitrogen gas, thus gaining access to a large “inert” pool. Many oceanic eucaryotic phytoplankton have reduced
their dependence on Fe by evolving a biochemical composition
that needs considerably less Fe. Of critical importance is that
no organisms are capable of reducing their
dependence on both combined N and Fe.
Cyanobacteria that fix N have high Fe requirements, and eucaryotes with low Fe re-
quirements cannot fix N2. Both groups are
needed to overcome N and Fe limitation of
new production and elevate productivity
to
a level that forces PO4 to become the ultimate limiting nutrient of biomass on a global basis.
,4lthough NO3 is depleted slightly before
PO4 in most areas of the ocean (Redfield
19 5 8; Fanning 19 89) Redfield correctly
pointed out that N cannot be the ultimate
limiting nutrient on a geochemical time scale
because of the large atmospheric pool of
molecular N accessible through N2 fixation.
Fe availability does appear to influence the
biomass and global distribution of N,-fixing
cyanobacteria. It appears that eucaryotic
phytoplankton
are not Fe limited directly,
but they could be indirectly limited because
of their ultimate dependence on N2 fixation
(w:hich is Fe limited) to counteract the effects of denitrification.
The critical factor is
that in this role, Fe is acting as a catalyst
and not limiting biomass directly. Although
Fe availability appears to limit the biomass
and control the distribution
of cyanobacteria, it does catalyze enough N2 fixation to
keep denitrification
from depleting the
ocean. The fact that NO3 is not depleted
from surGace waters until most of the PO4
is also depleted is evidence that N,-fixation
rates are sufficient to prevent N from becoming the geochemically limiting nutrient.
Th.e fact that N2 fixation has been able to
keep up with denitrification
on a global scale
is evidence that N,-fixation
rates are controlled by a feedback mechanism rather than
by some basic geochemical constraint such
as Fe. At most, only a small percentage of
the production of new biomass is limited
by the small depletion of N relative to P by
denitrification.
To the extent that Fe limitation prevents N2 fixation from eliminating
this N deficit completely, one could argue
that a small fraction of new production is
ultimately Fe limited.
N, P, and Fe can each be considered to
be limiting new production of biomass of
certain components of the phytoplankton
community. Because of the relationship between procaryotes and eucaryotes and a variety of complex biological interactions that
vary over time and space and alter species
composition, any of the three nutrients can
Minimum
iron requirements
end up limiting the maximum amount of
biomass produced locally on an ecological
time scale. Both NO3 (Eppley 198 1) and Fe
(Martin and Fitzwater 1988; Martin et al.
1989; Martin et al. 1990a) have been shown
to limit the growth rates of phytoplankton
in the ocean, but a nutrient that limits growth
rate does not necessarily limit the final yield
of biomass produced from the complete depletion of nutrients. It appears that on an
evolutionary
and geochemical time scale,
the total amount of new biomass that can
be produced globally from new nutrients entering the photic zone of the ocean is limited
by PO+ The addition of NO3 or Fe would
be expected to result in a species shift but
not significantly more biomass.
Conclusions
Rather large differences exist among marine phytoplankton
in their minimum
Fe
requirements. In the open ocean, eucaryotic
algae seem to need only a hundredth as much
Fe as cyanobacteria. The fact that oceanic
eucaryotes need much less Fe than coastal
eucaryotes is evidence that Fe has exerted
selective pressure in the ocean and that oceanic eucaryotes have been able to evolve a
biochemical composition that reduces the
problem of Fe scarcity. It appears that the
cyanobacteria have been unable to evolve
a significantly lower Fe requirement. A rough
comparison of the minimum Fe : P molar
ratio requirements of various phytoplankton species with the estimated fluxes of Fe
and PO4 to the photic zone from deep water
and the atmosphere suggests that new production of eucaryotic biomass would not be
Fe limited but production of cyanobacterial
biomass could be in parts of the ocean, particularly where atmospheric input is low
compared to deep-water input and where
dissolved Fe : P ratios in the deep water are
lowest.
The global and vertical distributions
of
cyanobacteria are consistent with the patterns of Fe and PO4 input to the photic zone
as a result of the high Fe : P ratios of atmospheric inputs and low Fe : P ratios of
deep-water inputs. Cyanobacteria seem to
be a large fraction of the phytoplankton
community
in the upper photic zone of
stratified waters where the ratio of atmo-
1769
spheric to deep-water input is high, a smaller fraction in the lower photic zone where
the ratio of atmospheric to deep-water input
is lower, and quite sparse in the Southern
Ocean where the ratio is very low. N,-fixing
Trichodesmium also appears to be most
abundant where the atmospheric input of
Fe is high compared to the deep-water input. It seems that N, P, and Fe availability
all influence species composition of the phytoplankton
community,
but at the same
time, the complex biological interactions
that determine species composition can also
influence the degree of nutrient limitation
of new production in the community by each
of the nutrients because of the large differences among species in their nutrient requirements. As a result, all three nutrients
can end up as important limiting nutrients
on an ecological time scale at various times
and places, but at least some species have
evolved adaptations that reduce or eliminate their need for combined N or Fe. Because no species have evolved a mechanism
for reducing their dependence on scarce P04,
it is concluded that PO, is the ultimate limiting nutrient of new production of organic
C on a geochemical and evolutionary time
scale.
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