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The connection of the phosphorus cycle to diazotrophs and nitrogen fixation
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The connection of the phosphorus cycle to diazotrophs and nitrogen fixation
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Content
THE CONNECTION OF THE PHOSPHORUS CYCLE TO DIAZOTROPHS AND
NITROGEN FIXATION
by
Jill Autumn Sohm
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOLOGY)
December 2007
Copyright 2007 Jill Autumn Sohm
ii
DEDICATION
To Adi, for supporting me and my work schedule during the last 5 years, my parents,
for never thinking I was crazy for wanting to do this, and Doug, for guiding me
while letting me find my own way.
iii
ACKNOWLEDGEMENTS
I would like to thank the captains and crew of the Research Vessels Seward Johnson,
Roger Revelle, and Endeavor, Margaret Mulholland, Gregory Cutter, Joseph
Montoya, and Jonathan Zehr who graciously invited me on their research cruises,
and David Karl, Karin Björkman, Paul Morris, Sergio Sanudo-Wilhelmy, Adam
Kustka, Edward Carpenter, George Boneillo, and Joseph Montoya for providing
various nutrient, count and chlorophyll data. This work was supported by NSF
grants OCE 99 81545, OCE 99 81371, and OCE 04 52765 to Douglas G. Capone.
Travel for work in Chapter IV was funded though a Gordon and Betty Moore
Foundation grant to John Zehr.
iv
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT ix
CHAPTER I: Limitation of nitrogen fixation in the marine environment:
The importance of marine nitrogen fixation and its control by phosphorus
and iron 1
INTRODUCTION 1
DISSERTATION AIMS 22
CHAPTER I REFERENCES 25
CHAPTER II: Assessment of Relative Phosphorus Limitation of
Trichodesmium in the North Pacific and Atlantic and the North Coast
of Australia 34
CHAPTER II ABSTRACT 34
INTRODUCTION 35
MATERIALS AND METHODS 38
RESULTS 43
DISCUSSION 49
CHAPTER II REFERENCES 57
CHAPTER III: Phosphorus dynamics of the tropical and subtropical north
Atlantic: Trichodesmium vs. bulk plankton 61
CHAPTER III ABSTRACT 61
INTRODUCTION 62
MATERIALS AND METHODS 64
RESULTS 69
DISCUSSION 73
CHAPTER III REFERENCES 83
v
CHAPTER IV: Phosphorus pools and cycling in the tropical North Atlantic:
Intrabasin differences and the influence of nitrogen fixation 87
CHAPTER IV ABSTRACT 87
INTRODUCTION 88
MATERIALS AND METHODS 91
RESULTS 94
DISCUSSION 100
CHAPTER IV REFERENCES 112
CHAPTER V: The connection of the phosphorus cycle to diazotrophs and
nitrogen fixation 116
INTRODUCTION 116
SUMMARY OF RESULTS 116
SYNTHESIS 120
CHAPTER V REFERENCES 131
ALPHABETIZED BIBLIOGRAPHY 134
vi
LIST OF TABLES
Table 2-1: V
max
and APA at station in the North Atlantic, North Pacific 45
and north of Australia.
Table 2-2: Summary of PO
4
3-
uptake kinetics and APA, surface nutrient 46
concentrations, and Trichodesmium colony information in the North
Atlantic, North Pacific and north of Australia, shown with standard error in
parentheses.
Table 3-1: Concentrations of chl a, inorganic and organic phosphorus in the 70
North Atlantic Ocean and inorganic P uptake and alkaline phosphatase
activity of bulk water samples and Trichodesmium colonies at ambient DIP
and DOP concentrations.
Table 3-2: V
max
, K
s
, and α for bulk water and Trichodesmium samples. 73
Table 3-3: Average volumetric rates of inorganic P uptake and alkaline 74
phosphatase activity for bulk plankton and Trichodesmium, assuming 1
colony per liter of water (Carpenter et al. 2004), and the percentage of total
uptake or activity each group is responsible for.
Table 4-1: Phosphorus pool concentrations in surface waters of different 101
regions of the ocean, as reported in previous studies.
Table 4-2: PO
4
3-
turnover times in surface waters of different regions of the 104
ocean, as reported in previous studies.
Table 4-3: The relationship of PO
4
3-
turnover times with affinity for PO
4
3-
( α). 109
vii
LIST OF FIGURES
Figure 1-1: Images of Trichodesmium colonies in the common tuft (left) 5
and puff (right) formations.
Figure 1-2: Image of unicellular cyanobacteria enriched from the North 8
Pacific in nitrogen free media.
Figure 2-1: Location of stations where Vmax ( z ) and APA ( ×) were 38
measured on the three cruises in this study.
Figure 2-2: PO
4
3-
uptake in 50% of surface light versus the dark for colonies 43
collected in the north Atlantic.
Figure 2-3: Per colony PO
4
3-
uptake rate measured throughout the day 44
(sunrise to sunset) in the north Atlantic and Pacific.
Figure 2-4: Examples of PO
4
3-
uptake kinetic curves from the North Atlantic 47
(top panel), North Pacific (middle panel) and waters north of Australia
(bottom panel).
Figure 3-1: A map of the area studied in the subtropical and tropical North 65
Atlantic showing station locations.
Figure 3-2: Examples of kinetic curves for bulk water samples (a) and 72
Trichodesmium colonies (b).
Figure 4-1: Salinity of surface waters in the tropical North Atlantic, shown 95
in units of psu.
Figure 4-2: Surface water P pool concentrations, in nM, in the tropical North 96
Atlantic: a) SRP, b) POP and c) DOP.
Figure 4-3: The relationship of SRP to POP in surface waters of the tropical 97
North Atlantic for all stations where both measurements were made.
Figure 4-4: Distribution of P in the SRP, DOP, and POP pools in surface 98
waters of thee tropical North Atlantic.
viii
Figure 4-5: The longitudinal gradient of PO
4
3-
turnover times in surface 99
waters of the tropical North Atlantic.
Figure 4-6: The longitudinal gradient in maximal PO
4
3-
uptake rates in the 100
globally significant diazotroph, Trichodesmium spp. in the tropical North
Atlantic.
Figure 4-7: The longitudinal change in DIN:SRP in the tropical North 106
Atlantic at 130-160 m ( z ) and at 200 m ( { ).
ix
ABSTRACT
Nitrogen (N
2
) fixation is an important component of the marine nitrogen (N) cycle,
as it provides a major source of new N to the oligotrophic ocean, which often N
limited. This input could increase global N inventories, leading to the sequestration
of atmospheric CO
2
via the biological pump. Any changes in N
2
fixation can
therefore theoretically change the carbon (C) sequestration capacity of the ocean. N
2
fixing organisms, like the filamentous cyanobacterium Trichodesmium spp., are
inherently not N limited. One would thus expect diazotrophs to thrive in the
oligotrophic oceans; this is not always the case. Something must be limiting N
2
fixation, and both phosphorus (P) and iron (Fe) are possible limiting nutrients.
However it is not yet clear which nutrient limits N
2
fixers in different locations.
With this in mind, a study was undertaken to understand where P can be limiting to
Trichodesmium spp. and the relationship between the P cycle and N
2
fixers.
Two assays used as indices of P limitation in Trichodesmium spp. are uptake of
33
PO
4
3-
to determine maximal P uptake (V
max
) and hydrolysis of P from
methylumbelliferone-phosphate to estimate alkaline phosphatase activity (APA).
The kinetics of PO
4
3-
uptake were determined for Trichodesmium spp. colonies in
the North Pacific, North Atlantic and in waters north of Australia while APA was
determined in the North Pacific and North Atlantic. Trichodesmium spp. V
max
was
x
significantly greater (~4 fold or more) in the North Atlantic compared to the North
Pacific and waters north of Australia when normalized to both Chl a content and
number of trichomes per colony. APA in the North Atlantic was also greater than in
the North Pacific. Our results indicate that Trichodesmium spp. is more strongly P
limited in the North Atlantic compared to the North Pacific or waters along the north
coast of Australia. Because P stress is important in the North Atlantic and
Trichodesmium spp. colonies are very large, the ability of Trichodesmium spp. to
compete for and acquire P is extremely important there. Nano- and pico-plankton
present in bulk water samples have a half saturation constant (K
s
) for PO
4
3-
nearly 30
times lower than that of Trichodesmium spp and account for >99% of PO
4
3-
uptake
in these waters. However, while chl a normalized alkaline phosphatase activity
(APA) in bulk water was an order of magnitude greater than in Trichodesmium spp.,
Trichodesmium contributes substantially to total APA in the water. Trichodesmium
is outcompeted for inorganic P, but colonies can satisfy their P needs by
supplementing PO
4
3-
uptake with P cleaved from DOP via alkaline phosphatase.
Having shown that the Trichodesmium spp. in the North Atlantic are P stressed and
must exploit the DOP pool to compete with nano- and picoplankton, the question
remains if the rest of the osmotroph community is P stressed in the North Atlantic,
and what the spatial pattern is over the entire basin. Despite the fact that SRP is
elevated in the Amazon River plume in the far western North Atlantic compared to
the rest of the basin, PO
4
3-
turnover times, considered an indicator of P stress,
xi
increase from <10h (more stressed) in the west to >100h (less stressed) in the east.
This could be due to surface water enrichment in NO
3
-
relative to PO
4
3-
above the
Redfield ratio of 16:1. Indeed, DIN:SRP ratios in subsurface waters were ~60 in the
western basin and decreased to ~16 in the eastern basin. N enrichment without a
concurrent enrichment in P may be a result of N
2
fixation, and published data does
indicate that Trichodesmium spp. is more abundant and active in the western basin
than the eastern basin. Trichodesmium spp. V
max
of PO
4
3-
uptake was also high in
the west and low in the east, indicating that, by enriching waters in N relative to P,
this diazotroph is increasing the competition for P between all organisms of the
osmotrophic community, including themselves.
1
CHAPTER I: Limitation of nitrogen fixation in the marine environment:
The importance of marine nitrogen fixation and its control by phosphorus and iron
INTRODUCTION
The marine environment has a fixed nitrogen (N) inventory of 8 × 10
5
Tg N (Libes
1992). This pool turns over about once every 3,000 years and is controlled mainly
by biological processes carried out by microbes (Codispoti 1995). The major inputs
of N to the ocean are atmospheric deposition, riverine input and N
2
fixation, the
dominant process being N
2
fixation. The major losses of N are burial and microbial
conversion of N to N
2
gas, either by respiratory denitrification or anaerobic
ammonium oxidation, with microbial processes apparently dominating losses
(Gruber and Sarmiento 1997, Kuypers et al. 2005). N
2
fixation is found in both the
water column and in sediments, and is classically thought to occur where fixed N is
low. However, recent unpublished evidence shows it can occur where fixed N is
present in the water column (below the thermocline in the San Pedro Channel; D. G.
Capone pers. comm.) or in sediment pore waters (in bioturbated sediments of
Catalina Harbor; V. Bertics pers. comm.).
The ocean is generally thought to be N limited, as this is the most abundant
macronutrient in phytoplankton, and in many marine ecosystems, some phosphorus
2
(P) remains when N is completely depleted (Ryther & Dunstan, 1971, Thomas,
1966). However, it is often argued thst P is limiting over geological timescales, as
N
2
fixation can replenish any N deficiencies in the ocean (Froehlich 1984, Tyrell
1999). N
2
fixing organisms occur in low nutrient, low chlorophyll waters (LNLC)
where N is depleted. Diazotrophs are successful in these areas because they are
inherently not N limited. They can take advantage of the largest reservoir of N on
the planet, N
2
gas, which other organisms cannot exploit. Due to this ability, one
would expect to find them growing abundantly in low nutrient areas, yet they
generally are not. Globally, something is controlling the growth of diazotrophs;
most likely it is other limiting nutrients. P and iron (Fe) are likely candidates for
limiting diazotrophic growth. P is an important macronutrient that is a part of,
among other things, energy-bearing molecules (ATP), protein synthesis machinery
(ribosomal RNA), and genetic material (DNA and RNA) and the Fe requirement of
diazotrophs is greater than bulk phytoplankton due to its presence in nitrogenase, the
enzyme that catalyzes N
2
fixation (Kustka et al. 2003, Raven 1988). It is not known
which, or if, one nutrient is more important in limiting N
2
fixation, however it is
likely that either nutrient may be limiting depending on the area of the ocean one is
studying.
N
2
fixation is an important biogeochemical process linked to C sequestration through
the biological pump. Many years ago, it was recognized that over the long term,
3
primary production that is removed to the deep ocean (export production) is in
balance with “new” production (production from new N), as opposed to N recycled
in the euphotic zone (Dugdale and Goering 1967, Eppley and Peterson 1979). For
years, upwelled nitrate was considered the only important source of new N to the
euphotic zone. New estimates, however, suggest that N
2
fixation is an important
source as well (Capone et al. 1996, Gruber and Sarmiento 1997, Michaels et al.
1996, Michaels et al. 2001, Capone et al. 2005). Considering that excess CO
2
is
brought up with nitrate from below the thermocline, N
2
fixation, in addition to
atmospheric deposition, are the only sources of truly “new” N that can effect a net
sequestration of CO
2
from the atmosphere to deep waters, via the biological pump.
The amount of new N in the ocean controls the strength of the biological pump. As
the oceanic N budget currently appears balanced (Gruber and Sarmiento 1997,
Gruber 2004), with inputs equaling outputs, C sequestration cannot change.
However, if the N cycle were thrown out of balance – N
2
fixation increased for
example – C sequestration in the ocean could increase. The implications of changes
in N
2
fixation on climate change are clear, due to the direct importance of N
2
fixation
to C sequestration in the ocean. The study of what controls N
2
fixation, how
diazotrophs acquire limiting nutrients, and how we recognize nutrient limitation
therefore becomes important.
4
N
2
-fixation: N
2
fixation, the process of converting N gas into ammonia, is carried
out exclusively by prokaryotes called diazotrophs. The reaction is catalyzed by the
enzyme nitrogenase which consists of two proteins, one containing Fe atoms and the
other containing both Fe and molybdenum. The number of Fe atoms per molecule
varies by species, but lies in the range of 20-35 for the large subunit and 4 for the
small subunit. The synthesis of nitrogenase is controlled by the nif operon. The
reduction of N
2
to two molecules of NH
3
is a costly one, consuming 16 ATP
molecules in the process. This number is the cell-free need of nitrogenase; the actual
ATP use efficiency varies by species. Reducing power for nitrogenase comes from
ferredoxins and flavodoxins. Which molecule is used, and how, depends on the
species.
Nitrogenase is extremely oxygen sensitive, a problem that different N
2
fixers solve in
different ways. Some organisms, such as some of the cyanobacteria, spatially
separate nitrogenase from oxygen in a heterocyst, and others, like unicellular
cyanobacteria, temporally segregate oxygenic photosynthesis from N
2
fixation by
fixing N
2
at night when oxygen levels are lower. N
2
fixation occurs in many
different physiological types of prokaryotes: anaerobes, facultative anaerobes,
aerobes, and phototrophs. Yet the ability to carry out N
2
fixation is rare, perhaps
restricted to a few hundred prokaryotes (Postgate 1998).
5
Trichodesmium: Trichodesmium is a colony forming cyanobacteria that is
cosmopolitan in oligotrophic tropical and subtropical waters (see figure 1-1, Capone
et al. 1997). High abundances are seen in waters with very stable water columns and
deep mixed layers (100m) such as western boundary currents, tropical central gyres
and some ocean margin seas (Capone et al. 1997). Surface blooms of
Trichodesmium that are visible to the eye are fairly common in all these areas,
especially when wind speeds are low and water temperature is above 25
o
C
(Carpenter and Capone 1992).
Figure 1-1 Images of Trichodesmium colonies in the common tuft (left) and
puff (right) formations. Courtesy of John Waterbury at the Woods Hole
Oceanographic Institute.
Trichodesmium is a nonheterocystous cyanobacteria. Interestingly, it fixes N
2
in the
daylight while simultaneously evolving O
2
, despite the fact that nitrogenase is
extremely sensitive to oxygen. In fact, N
2
fixation in Trichodesmium follows an
endogenous rhythm, and will not fix N
2
even when placed in artificial light during
the night (Saino and Hattori 1978, Capone et al. 1990). Trichodesmium can take the
6
form of colonies or free filaments; in most areas colonies are the main morphology,
but in the North Pacific Subtropical Gyre free filaments are a significant portion of
the biomass (Letelier and Karl 1996). Free filaments have a lower Chl a-specific N
2
fixation rate than colonies (Letelier and Karl 1998). Growth rates of Trichodesmium
in the field are relatively low compared to eukaryotes (Carpenter 1983) and doubling
times in culture average 3-5 days.
The biogeochemical significance of Trichodesmium has only been recognized
relatively recently. Due to poor sampling technique, the density and distribution
were previously underestimated. Use of better techniques led to an estimate of 80 Tg
N annual input by Trichodesmium in the waters that it occurs (Capone et al. 1997).
This amounts to about half the total estimated marine N
2
fixation (Gruber and
Sarmiento 1997). Still, this may be a conservative estimate, as it does not include the
input from blooms; they have not been studied extensively enough to estimate their
contribution. Additionally, the use of video plankton recorders indicates that
Trichodesmium is even more widespread in the North Atlantic than previously
thought (Davis and McGillicudy 2006). Continued use of this technology may
expand the known area and abundances of Trichodesmium.
Trichodesmium is responsible for a large input of fixed N to the oceans, however, it
is not altogether clear how the N
2
fixed by Trichodesmium is made available to other
7
organisms. It is known that some of the fixed N is released by cells as amino acids
and organic matter (DON) (Capone et al. 1994, Gilbert and Bronk 1994), and that
viral lysis may be another important mechanism of N release (Hewson et al. 2004).
Recent evidence shows that N
2
fixation does not contribute to the measurable DON
pool in the oligotrophic ocean (Knapp et al. 2005, Meador et al. 2007) but likely
contributes to a rapidly cycling, and thus undetectable, DON pool that is important to
free living bacteria (Meador et al. 2007). In addition to the large new N input that
Trichodesmium appears to provide to the waters it inhabits, it may also be a
significant portion of the biomass in a given body of water, contributing considerably
to primary productivity (Carpenter et al. 2004).
Picoplankton: Not much is currently known about the role picoplankton play in
global N
2
fixation, as much attention has been paid over the years to Trichodesmium.
However, N
2
fixation by Trichodesmium alone cannot account for all the excess N
found in some areas of the ocean. Less than a decade ago, N
2
fixation in the pico-
plankton fraction was not considered important and the diversity of N
2
fixers was
unknown. In 1998, Zehr et al. amplified over 30 different nifH genes from the North
Pacific subtropical Gyre and the Sargasso sea related to cyanobacteria,
proteobacteria and sulfate reducers (see figure 1-2). nifH codes for one of the
subunits of nitrogenase.
8
Figure 1-2 Image of unicellular cyanobacteria enriched from the North Pacific in
nitrogen free media. From Zehr et al. (2001).
Zehr et al. (2001) subsequently showed expression of nifH genes in the NPSG and
N
2
fixation in cultured isolates, confirming that N
2
fixation in the picoplanktonic
fraction is occuring. Since these original studies, many researchers have amplified
nifH genes in diverse locations such as the North Pacific and Atlantic (Falcon et al.
2002, Falcon et al. 2004), the South China Sea (Chou et al. 2006), the Eastern
Mediterranean (Mar-Aharonivich et al. 2007) and deep waters of the Sargasso Sea
(Hewson et al. 2007). Other studies have shown expression of the nifH gene in the
North Pacific (Church et al. 2005a) and quantitative PCR methods have been
developed to quantify the abundance of picoplanktonic diazotrophs (using DNA nifH
copy number, Church et al. 2005a) and the abundance of nifH transcripts (using
reverse transcriptase PCR, Church et al. 2005b). This research has shown that the
unicellular cyanobacteria are the dominant producer of nifH transcripts in the
picoplankton, and thus are likely the dominant N
2
fixer. These methods, however, do
not allow for the estimation of actual N
2
fixation rates. A few studies have
9
quantified the N
2
fixation rates of the <10 µm fraction and found that areal rates can
sometimes equal or exceed measurements of Trichodesmium N
2
fixation. N
2
fixation
in the Eastern North Pacific gyre was found to be very high on one cruise, 520 µmol
N m
-2
d
-1
on average (Montoya et al. 2004), which is more than twice the areal N
2
fixation rate calculated for Trichodesmium in this area (Doug Capone unpublished
data). North of Australia, N
2
fixation in the <10 micron fraction averaged 126 µmol
N m
-2
d
-1
(Montoya et al. 2004), while in a warm core and cold core eddy off the
west coast of Australia, N
2
fixation was 1/3 and 1/6 of this value, respectively (Holl
et al. 2007). In the western Mediterranean Sea, areal rates ranged from 40-100 µmol
N m
-2
d
-1
(Garcia et al. 2006). Thus, unicellular cyanobacteria appear to be present
and fixing N
2
in many areas of the ocean. These picoplanktonic diazotrophs appear
to be able to support about 10% of oceanic new production, based on the estimates of
Lee et al. (2002), and their new N input is sometimes equal to or greater than
calculated estimates of deepwater nitrate diffusion or upwelling (Montoya et al.
2004). In addition, it appears that picoplanktonic organisms can be very important
locally to total N
2
fixation, as it can sometimes be responsible for nearly all the N
2
fixation measured in Trichodemium colonies and free filaments, diazotrophic
symbionts of diatoms, and picoplankton at some stations in the North Atlantic, North
of Australia, and in the North Pacific Subtropical Gyre (D. G. Capone, unpublished
data). Researchers are still studying what unicellular cyanobacteria exist in the field
and what their biogeochemical significance is in the oligotrophic oceans. Due to
10
their probable significance, it is important to look at what controls their N
2
fixation
rates, as it may be different from Trichodesmium. Because the picoplanktonic
diazotrophs cannot be separated from other organisms in the field, new methods
must be developed to study nutrient limitation of these organisms. Molecular
markers for one group of unicellular cyanobacteria are in development (Eric Webb,
personal communication), but another approach is to do nutrient addition
experiments and look for an increase in transcription of the nifH gene over the course
of an incubation. Zehr et al. (2007) carried out a PO
4
3-
addition to the <10 micron
fraction of water from Kaneohe Bay, Hawaii and found that the addition did not
elicit an increase in nifH expression. This would suggest that unicellular diazotrophs
from this location were not P limited. In order to assess what is limiting to
picoplanktonic diazotrophs, especially the unicellular cyanobacteria, more bioassays
of this type need to be carried out.
Iron limitation: Over 15 years ago, John Martin (1990) hypothesized that
phytoplankton production in high nutrient low chlorophyll (HNLC) waters such as
the equatorial Pacific and Southern Ocean was Fe limited. Large-scale fertilization
projects like IronExII and SOIREE showed this hypothesis to be correct (Boyd et al.
2000, Coale et al. 1996). Coastal areas have also been shown to be Fe limited
(Hutchins and Bruland 1998). Due to the high Fe requirement of N
2
fixation and low
atmospheric dust deposition to areas of N
2
fixation (low nutrient low chlorophyll, or
11
LNLC areas), some hypothesize that Fe limits N
2
fixation globally (Falkowski 1997,
Rueter et al. 1992). However, a new calculation estimates that Fe requirements of N
2
fixing phytoplankton are only 2.5-5.2 times higher than for NH
4
+
- assimilating
phytoplankton (Sañudo-Wilhelmy et al. 2001), much less than previous estimates of
100 times higher (Raven 1988). Culture experiments with Trichodesmium confirm
that the Fe requirement for moderately Fe limited growth is indeed five fold greater
for diazotrophy compared to ammonium-based growth (Kustka et al. 2003a). This
difference, however, is likely still important in the waters where N
2
fixation occurs,
as dissolved Fe levels are generally <1nM (Sañudo-Wilhelmy et al. 2001).
Fe limitation and uptake in Trichodesmium and other cyanobacteria has been
relatively well studied in recent years. In 1988, Rueter found increases in CO
2
and
N
2
fixation rates and cellular chlorophyll a content in natural samples of
Trichodesmium collected near Barbados and amended with Fe. More recently, N
2
fixation rates in Trichodesmium cultures were shown to be positively correlated to
cellular Fe quotas (Berman-Frank et al. 2001). Combining this data and data on
aeolian dust fluxes, Berman-Frank et al. (2001) estimate that N
2
fixation is Fe limited
in 75% of the world’s oceans. Evidence for dust stimulation of Trichodesmium
biomass has recently been reported. A 1999 Saharan dust event coincided with
increases in dissolved Fe concentrations on the West Florida shelf and a 100-fold
increase in Trichodesmium biomass. N
2
fixation rates were not measured, but DON
12
concentrations did increase in the water over background concentrations (Lenes et al.
2001).
Trichodesmium cultures show increases in Fe:C when supplied with increasing
amounts of Fe. It was subsequently shown that carbon-specific N
2
fixation rates in
field populations increased with the Fe:C ratio in Australia, where it appears
Trichodesmium is not limited by P, but not in the Atlantic, were they are P limited
(see below). In addition, N
2
fixation in colonies collected north of Australia was
stimulated by Fe additions (Kustka et al. 2003b). Thus, in waters north of Australia,
N
2
fixation by Trichodesmium appears Fe limited. Experiments on a Trichodesmium
isolate from the Great Barrier Reef lagoon grown at a range of Fe concentrations
indicate that N
2
fixation in Trichodesmium is more sensitive to Fe limitation than cell
growth (Fu and Bell 2003). The bioavailability of this potentially limiting nutrient to
Trichodesmium is an important aspect to Fe limitation. A study of field collected
samples showed that Trichodesmium could take up Fe in both the inorganic Fe(III)
form and from a siderophore at similar rates, while other Fe-binding ligands
decreased availability (Achilles et al. 2003).
Phosphorus limitation: There is no analog to N
2
fixation for P. P is only input to the
ocean by weathering of PO
4
3-
off the continent and riverine discharge. This suggests
that the ocean is P limited on a geological time scale; N
2
fixation will increase when
13
N becomes limiting and drive the ocean to P limitation (Tyrell 1999). Still, marine
phytoplankton are generally thought to be N limited, while diazotrophs clearly are
not. The theory of long-term P limitation in the ocean implies that P supply controls
the global rate of N
2
fixation. Considering the importance of P in phytoplankton,
this may well be true.
Although it is acknowledged that the ocean is N limited in the short term and P
limited in the long term, there are many areas of the ocean that are currently P
limited. Alkaline phosphatase activity and P turnover times show large areas of the
subtropical Atlantic and Sargasso Sea are P limited (Cotner et al. 1997, Rivkin et al.
1997, Vidal et al. 2003). A study of inorganic P and dissolved Fe distributions also
suggest this area is P limited (Wu et al. 2000). The Mediterranean also appears P
limited, due to high nitrate to phosphate ratios (Krom et al. 1991) and extremely fast
turnover times of the DIP pool (Thingstad et al. 1998). In addition, APA and P
uptake indicate stronger limitation in the Eastern Mediterranean Sea (Zohary and
Robarts 1998). Very recently, researchers have found P limitation of
bacterioplankton, ciliates and copepod egg production, and P and N co-limitation of
picophytoplankton through large scale addition of P to a patch of Eastern
Mediterranean water (Thingstad et al. 2005). Transient P limitation is found in the
summer in the Baltic Sea, when blooms of Nodualria and Aphanizomenon are
present (Moisander et al. 2003, Nausch et al. 2004). The presence of APA in the
14
mixed layer of the central North Pacific led Perry (1972) to suggest P defiency of
phytoplankton in this area. Additionally, a selection for N
2
fixing organisms in the
North Pacific Subtropical Gyre at station ALOHA suggests this area is in a transition
from N to P limitation (Karl and Tien 1997). However, a different study asserts this
area is Fe, rather than P, limited (Wu et al. 2000).
There is not much published data on the role of P in controlling N
2
fixation in the
open ocean. Samples of water from the Baltic Sea consistently showed higher
nitrogen fixation rates at stations where DIP was in excess relative to DIN (i.e. <16:1
ratio) compared to stations where DIP was depleted relative to DIN. In addition, a
weak linear correlation between P supply and N
2
fixation rates was observed,
indicating P limitation of diazotrophs in the Baltic (Moisander et al. 2003). Both Fe
and P additions to water from the eastern tropical North Atlantic stimulated N
2
fixation, showing that in this area, N
2
fixation is co-limited by Fe and P (Mills et al.
2004). Sañudo-Wilhelmy et al. (2001) found that N
2
fixation in Trichodesmium
from the central Atlantic Ocean was tightly correlated to P content of the colonies,
suggesting P limitation of N
2
fixation in this area. Inorganic P is not the only P
source for Trichodesmium in the open ocean. Trichodesmium produces alkaline
phosphatase, which cleaves PO
4
3-
off of dissolved organic P compounds to provide
an additional P source to the organism. Alkaline phosphatase activity (APA) in
natural populations of Trichodesmium can indicate P stress to some degree, as it was
15
found that APA was much higher in the North Atlantic, where inorganic P
concentrations are extremely low, than off the northern coast of Australia where
concentrations are perhaps 10-fold higher (Mullholland et al. 2002). APA was also
found to be higher (10-fold) in P-deplete cultures of Trichodesmium strain WH9601
compared to P-replete cultures (Stihl et al. 2001). However, APA is not a direct
indicator of P limitation, but an indication that Trichodesmium are experiencing low
levels of inorganic P (Scanlan and Wilson 1999).
Indices of P Limitation and P Uptake Kinetics: Indices of P limitation includes the
presence of stress proteins or increase in the activity of these enzymes (e.g. alkaline
phosphatase), Chl:C and N:P ratio, or the response of physiological parameters such
as N
2
fixation rate to P additions. Michaelis-Menten nutrient uptake parameters are
also known to vary with nutrient stress in many organisms (Gotham and Rhee 1981,
Donald et al. 1997, Ikeya et al. 1997, Riegman et al. 2000).
Elemental ratios in phytoplankton have long been assumed to be relatively fixed,
particularly by members of the geochemical community. Some have questioned this
view (Falkowski 2000), and indeed it has been shown that ratios can vary locally and
with nutrient limitation (Geider & LaRoche 2002). Evidence from the field and
cultures suggests that the N:P of Trichodesmium biomass is highly dynamic and that
P limitation increases these ratios over relatively short time scales (Krauk 2000,
16
Krauk et al. 2006). In addition, it has also been shown in the North Atlantic, a
system that appears to be strongly P limited, that P content rather than Fe content is
related to N
2
fixation rates by Trichodesmium (Sañudo-Wilhelmy et al. 2001). It is
important to note, however, that elemental ratios are an indicator of Leibig limitation
rather than growth limitation of phytoplankton by a given nutrient at a specific point
in time, i.e. N:P ratios are a cumulative indicator of P stress and can be high even if
growth is limited by another nutrient (Beardall et al. 2001).
Alkaline phosphatase is an ectoenzyme that cleaves monophosphate ester bonds off
of dissolved organic compounds. Studies from many different environments and
many different organisms show that alkaline phosphatase activity (APA) in
phytoplankton increases when organisms are P starved (Hoppe 2002). APA can also
be an important source of P to cells. Alkaline phosphatase appears to be very
important to Trichodesmium. Turnover times of Trichodesmium due to alkaline
phosphatase in waters north of Australia were calculated to be 6 to 150 hours, which
means that APA can account for a significant amount of the P demand (Mulholland
2002). It appears then, that the APA of diazotrophs (for Trichodesmium at least) is
an important component to P cycling. Nutrient cycling by diazotrophs and nutrient
stress can also be measured by inorganic nutrient uptake.
17
Nutrient uptake kinetics can be described by the Michaelis-Menten equation:
V = (V
max
× S)/(K
s
+ S)
V is the uptake rate of a nutrient at concentration S, V
max
is the maximal, or
saturated, uptake rate, and K
s
is the nutrient concentration where V is one half V
max
(called the half saturation constant). It is well known that phytoplankton will increase
nutrient uptake ability in response to nutrient limitation. Graziano et al. (1996)
found a ten-fold increase in maximum P-uptake rates of P-limited Dunaliella
tertiolecta compared to P-replete cultures. This response was due to an increase in
uptake sites, as the half saturation constant was unchanged. Microcystis aeruginosa,
a freshwater cyanobacterium, adapted to P limitation by increasing its phosphate
uptake capacity and decreasing its light harvesting capacity (Kromkamp et al. 1989).
In culture experiments comparing the P nutrition of the marine cynaobacterium
Synechococcus with the diatom Thalassiosira weissflogii showed that P starved
Synechococcus increased V
max
almost 100 fold and K
s
decreased 20 fold compared to
P replete cultures, while P starved T. weissflogii only increased V
max
by an order of
magnitude over P replete cells, with K
s
not changing between the two treatments
(Donald et al. 1997). It was subsequently shown that, due to their great differences
in P uptake kinetics under P limitation, Synechococcus outcompetes T. weissflogii
under P deplete conditions, while T. weissflogii wins when P is abundant. With the
re-supply of limiting nutrients, cells exhibit uptake rates that are higher than needed
to maintain growth (Parslow, 1984), a phenomenon referred to as “luxury uptake.”
18
Increased PO
4
3-
uptake rates can also be seen in natural samples when P
concentrations are reduced. PO
4
3-
uptake rates for all size fractions measured on a
transect in the NE Atlantic were increased off the shelf where PO
4
3-
concentrations
were the lowest (Donald et al. 2001).
Three studies have so far been conducted on the P uptake kinetics of Trichodesmium.
McCarthy and Carpenter (1979) measured uptake for field populations at one station
in the Central North Atlantic between Bermuda and Spain and found an extremely
high K
s
of 9 µM. Fu et al. (2005) subsequently found a K
s
for cultured
Trichodesmium (IMS101) of about 0.4 µM for both P replete and deplete cells in
batch cultures. While K
s
did not change, V
max
increased by an order of magnitude in
P deplete cultures, versus P replete cultures, showing that V
max
has value as an
indicator of P stress in field populations. However, these values do not take into
account the possibility of multiphasic kinetics, and thus may suffer be influenced by
artifacts.
P acquisition by Trichodesmium: The question of how, exactly, a Trichodesmium
colony is able to acquire enough P in DIP deplete surface waters to survive,
especially in P limited areas, is a topic of some debate. Karl and colleagues (1992)
have suggested that colonies may ballast themselves and sink to the phosphocline,
where they take up P, then travel back to the surface to grow. Data on N:P ratios of
19
floating and sinking colonies at the surface and at depth from the North Pacific
(Letelier et al. 1998) and North of Australia (Villareal and Carpenter 2003) do not
support this hypothesis, while data from the Gulf of Mexico does (Villareal and
Carpenter 2003). Villareal and Carpenter (2003) undertook an extensive study of
carbohydrate ballasting in Trichodesmium and found that there is, in fact, a daily
vertical migration of Trichodesmium through the water column, with a majority of
colonies rising in the morning, and then sinking late in the day after spending time
near the surface and accumulating carbohydrates. However, based on the maximum
and minimum amounts of ballast found, the speed of migration measured, and an
estimation of the respiration rate of carbohydrates when colonies are in low light,
they calculated that a colony could sink a maximum of 55 m. If the colony started its
migration from 15 m, it could sink to 70 m at most. Considering that the
phosphocline in the waters where Trichodesmium is prevalent is usually greater than
100 m, it would be physically impossible for Trichodesmium to migrate to the
phosphocline to take up large amounts to P if Villareal and Carpenter (2003) are
correct. Ultrastructural investigation of rising and sinking Trichodesmium colonies
also suggests that they are getting their P from somewhere other than deep waters, as
polyphosphate storage granules are often seen in sinking colonies but not in rising
colonies (Romans and Carpenter 1994). Conversely, a recent model of
Trichodesmium ballasting and migration found that ~19% of simulations resulted in
colonies migrating to the depth of the phosphocline (White et al. 2006). Careful
20
field observations are needed to determine if P mining is actually happening.
Trichodesmium has been shown in cultures and field populations to exhibit alkaline
phosphates activity. If Trichodesmium can gain enough P from the DOP pool
through alkaline phosphatase activity, the mining of P from depth may not be
necessary to explain growth.
N
2
fixation and carbon sequestration: The ocean C pool is 60 times that of the
atmosphere (Martin 1990). Almost all of this pool is sequestered in the deep ocean,
and it is transported there by the sinking of cold surface water and by the export of
organic C from the euphotic zone. The magnitude of new production and export can
directly affect atmospheric CO
2
concentrations. During the last glacial maximum
(LGM) the concentration of CO
2
in the atmosphere was about 80 ppm lower than in
the pre-industrial interglacial period (Falkowski 2000). The consensus is that the
extra C was sequestered in the ocean (Martin 1990), but the mechanism by which
this occurred is unknown.
Geological evidence corroborates the theory that N
2
fixation may play a critical role
in accounting for increased CO
2
sequestration during glacial periods. Broeker and
Henderson (1998) argue that, due to the constraints of the events of Termination II,
the only hypothesis posited thus far that could account for the CO
2
change is a
decrease in the oceanic N inventory during deglaciation (i.e. a larger N pool was
21
responsible for the CO
2
drawdown in the last glacial maximum). Altabet et al.
(2002) and Ganeshram et al. (2000) have provided evidence that denitrification was
reduced during the last glacial maximum in the Arabian Sea and the eastern Pacific
margins (important sites of denitrification in today’s ocean), possibly increasing the
N inventory of the ocean. Ganeshram (2002) has more recently questioned this
hypothesis.
It has also been suggested that increases in aridity during the LGM could have
increased dust deposition to areas of N
2
fixation, increasing Fe delivery to Fe limited
diazotrophs. Supply of this limiting nutrient would have increased N
2
fixation, thus
increasing the N inventory of the ocean and the drawdown of atmospheric CO
2
(Hood et al. 2000, Falkowski 1997). Evidence indicates that dust flux to the
Southern Ocean increased by an order of magnitude in the LGM (Broeker and
Henderson 1998) but dust deposition on the rest of the world’s oceans during this
time is unknown. It is important to acknowledge that this scenario requires that
phytoplankton grew at an N:P ratio higher than the Redfield ratio of 16:1. Geider
and La Roche (2002) documented numerous examples of individual species growing
at ratios higher than 16:1.
While it may be that the change in the size of the nitrate pool was not the primary
driver of climate change, it likely was a factor. In addition, denitrification and N
2
22
fixation appear to be tightly coupled and feed back on each other relatively quickly
(Deutch et al 2004, Gruber 2004). If the N inventory of the ocean is decreasing, as
some assert it is (Codispoti et al. 2001) knowledge of the N
2
fixation response to
nutrient limitation is essential.
DISSERTATION AIMS
While it is acknowledged that both Fe and P could play an important role in
controlling N
2
fixation in the marine environment, the purpose of this research is to
investigate the relationship of P to diazotrophs and N
2
fixation. P limitation is
known to occur in some areas of the ocean, including the subtropical and tropical
North Atlantic (Cotner et al. 1997, Sanudo-Wilhelmy et al. 2001) and the
Mediterranean Sea (Thingstad et al. 2005), but there is disagreement over whether or
not the North Pacific Subtropical Gyre is one of these locations. Modeling studies
and investigation of dissolved nutrient concentrations suggest that N
2
fixation is P
limited in the North Atlantic and Fe limited in the North Pacific (Moore and Doney
2001, Wu et al. 2000), but a decadal scale decrease in SRP stocks in the North
Pacific led Karl and Tien (1997) to conclude that P must be limiting the entire
system. Bioassays to resolve this discrepancy have yet to be carried out.
23
Furthermore, colonies of Trichodesmium are very large for phytoplankton (>1 mm
across) and thus represent a concentrated package of P. Considering that SRP is
generally present in low amounts in surface waters of the oligotrophic ocean where
Trichodesmium occurs, and that, additionally, some of those areas might be P
limited, it is not clear how Trichodesmium is able to acquire all this P. Nano- and
pico-plankton, especially the sub-micron bacteria and cyanobacteria that would
dominate a small water sample, have very high surface area to volume ratios, and
thus, the ability to take up nutrients very rapidly at low concentrations. The lower
surface area to volume ratio of the large Trichodesmium colonies should diminish its
ability to take up PO
4
3-
and compete with nano- and pico-plankton for it. The DOP
pool, which is made up of monophosphate esters and phosphonates (Kolowith et al.
2001), could be an important source of P, as Trichodesmium possesses the genes
necessary to internalize P from both types of compounds (Orchard et al. 2003,
Dyhrman et al. 2006).
Finally, the long turnover time of the marine P pool and the slow geological source
and sink processes of weathering and burial of sediments have led many to ignore
the biological cycling of P. While it is not a dynamic cycle with multiple redox
transformations like the N cycle, it is clear that P is a biologically important nutrient
in P limited areas and to N
2
fixing organisms, and thus should not be overlooked. P
pools and PO
4
3-
cycling have been the subject of a few studies (for example: Tanaka
24
et al. 2003, Van Den Broeck et al. 2004,Yoshimura et al. 2007) but spatial variability
has not been well quantified, especially as it relates to areas with large amounts of N
2
fixation.
With these issues in mind, this research aims to determine the following: 1) if
Trichodesmium, an important marine diazotroph, shows differential P
limitation in different ocean basins based on some common P stress indices, 2) if
Trichodesmium can compete with nano- and pico-plankton for PO
4
3-
, and if not,
what pool do they get P from, and 3) how does P cycling and P pool composition
change in the tropical North Atlantic, in relation to areas known to be enriched
in diazotrophs.
25
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34
CHAPTER II: Assessment of Relative Phosphorus Limitation of Trichodesmium in
the North Pacific and Atlantic and the North Coast of Australia
CHAPTER II ABSTRACT
Trichodesmium spp. is a colonial diazotrophic cyanobacterium that occurs in the
oligotrophic tropics and subtropics. Because of its ability to fix atmospheric
dinitrogen (N
2
), it is likely to be growth limited by phosphorus (P) or iron (Fe), and it
has been hypothesized that limitation differs among different ocean basins. Two
assays used as indices of P limitation in Trichodesmium spp. are uptake of
33
PO
4
3-
to
determine maximal P uptake (V
max
) and hydrolysis of P from methylumbelliferone-
phosphate to estimate alkaline phosphatase activity (APA). The kinetics of PO
4
3-
uptake were determined for Trichodesmium spp. colonies in the North Pacific, North
Atlantic and in waters north of Australia, and APA was determined in the North
Pacific and North Atlantic. Trichodesmium spp. V
max
was significantly greater (~4
fold or more) in the North Atlantic compared to the North Pacific and waters north of
Australia; this was true when normalized to both Chl a content and number of
trichomes per colony. APA in the North Atlantic was also greater than in the North
Pacific. The half saturation constant for PO
4
3-
uptake (K
s
) was not significantly
different among the three locations. These results indicate that Trichodesmium spp.
is more strongly P limited in the North Atlantic compared to the North Pacific or
35
waters along the north coast of Australia. We suggest that the Trichodesmium spp.
communities in the North Pacific and waters north of Australia are primarily Fe
rather than P stressed and that these differences reflect differing relative inputs and
availability of two major controlling variables for diazotrophy, P and Fe, in these
geographically divergent areas.
INTRODUCTION
N
2
fixation is now recognized as an important source of new nitrogen (N) in the
tropical oligotrophic ocean (Gruber and Sarmiento 1997), and comparable to the
diffusive flux of nitrate from below the photic zone in those systems (Capone et al.
2005). However, where the upward flux of nitrate co-occurs with carbon dioxide
(CO
2
) at Redfield proportions, that nitrate cannot support a net flux of CO
2
from
atmosphere to ocean (Eppley and Peterson 1979; Michaels et al. 2001). Atmospheric
input of N or in situ N
2
fixation are required to promote positive carbon (C)
sequestration in the ocean. N
2
fixation in the ocean is thus inextricably linked to the
C cycle and possibly even climate change (Michaels et al. 2001). Understanding
what controls N
2
fixation in the open ocean is a vital part of understanding C cycling
in the past, present, and future ocean.
36
N has long been considered the primary limiting nutrient throughout the open ocean
(Thomas 1966). Diazotrophs avoid this limitation by exploiting the largest N
reservoir on the planet, N
2
gas, through the process of N
2
fixation. However, there is
no analog of this process for phosphorus (P). This led to the conclusion that P is the
ultimate limiting nutrient of marine primary production, as any N deficiencies are
made up by N
2
fixation (Tyrrell 1999). The assumption that follows is that marine
N
2
fixation is a P limited process. Alternatively, Fe may constrain diazotroph
growth, as diazotrophs have a higher Fe requirement than other phytoplankton (
Raven 1988; Kustka et al. 2003b), and Fe concentrations are generally low in the
upper ocean (Wu et al. 2001). This study focuses on the role of P in limiting open
ocean diazotrophs.
The colony forming, diazotrophic cyanobacterium Trichodesmium spp. is
cosmopolitan in tropical and subtropical waters and contributes a substantial amount
of new N to the areas where it occurs ( Capone et al. 1997; Capone et al. 2005).
Thus, the study of this one organism is important to our understanding of open ocean
N
2
fixation. Knowledge of Trichodesmium spp. susceptibility to P limitation in
different ocean basins will improve our understanding of its ability to contribute new
N to the open ocean.
37
Common indices used to diagnose P stress in phytoplankton are PO
4
3-
uptake
kinetics and the activity of alkaline phosphatase – an enzyme that cleaves PO
4
3-
from
organic P molecules. Many algal species respond to P stress by altering PO
4
3-
uptake
kinetics and alkaline phosphatase activity (APA) (for example: Donald et al. 1997),
and these measures can be used to infer relative P limitation (Beardall et al. 2001).
Recently, both maximal PO
4
3-
uptake (V
max
, (Fu et al. 2005)) and APA ( Stihl et al.
2001; Mulholland et al. 2002) were shown to increase with P depletion in
Trichodesmium IMS 101 cultures. Based on these culture studies, both V
max
and
APA can be used to compare relative P limitation in field populations of
Trichodesmium spp.
We undertook our study in three distinct regions predicted to have different
susceptibility to P limitation: the subtropical North Pacific where both P and Fe
limitation are hypothesized (Karl et al. 1997; Wu et al. 2000), the tropical North
Atlantic where P limitation of diazotrophs has been shown (Sañudo-Wilhelmy et al.
2001) and the north coast of Australia, where P concentrations are generally higher
than the two other areas. PO
4
3-
uptake kinetics and alkaline phosphatase activity
measurements were made to assess the relative P limitation of Trichodesmium spp.
colonies in these diverse locations. An additional set of experiments were carried
out to assess the diel periodicity and light dependence of Trichodesmium spp. PO
4
3-
uptake in the field.
38
MATERIALS AND METHODS
Study Sites: Data for this study were collected on three separate research cruises
(Figure 2-1).
Figure 2-1 Location of stations where Vmax ( z ) and APA ( ×) were
measured on the three cruises in this study.
39
The first cruise was in November 1999 aboard the R/V Maurice Ewing off the
northern coast of Australia, where Trichodesmium erythraeum was the predominant
species (E. J. Carpenter, pers. comm.). The second cruise was from April-May 2003
in the tropical North Atlantic, in and around the Amazon River plume, aboard the
R/V Seward Johnson. The third cruise took place from July-August 2003 in the
subtropical North Pacific, specifically the area south of the Hawaiian Islands, on the
R/V Roger Revelle. Trichodesmium thebautii was the dominant species on the North
Atlantic and Pacific cruises (E. J. Carpenter, pers. comm.).
33
PO
4
3-
uptake was
measured on all three cruises. APA measurements from the Pacific and Atlantic
cruises are presented in this study, while APA measurements from north of Australia
were previously presented in Mulholland et al. (2002). Because we encountered a
different predominant Trichodesmium species in Australia, we assume here that there
are not species specific differences in PO
4
3-
uptake and APA. Fu et al. (2005)
showed that uptake kinetics for PO
4
3-
in two different clonal isolates were the same
when size was considered, but differences between species for these parameters have
yet to be studied. To account for differences in colonies between the three sites,
kinetic and APA data are normalized to both colony chl content and trichome
number (provided by E. J. Carpenter – pers. comm.). Additionally, because
Trichodesmium spp. colonies support a community of heterotrophic bacteria,
cyanobacteria and other phytoplankton, among other things (O,Neil and Roman
1992), kinetic and APA measurements will include contributions from these
40
communities. It is possible that these organisms play a role in nutrient acquisition
and cycling within the Trichodesmium spp. colony. However, the total volume of
bacteria, diatoms and dinoflagellates found on colonies collected in the North
Atlantic was less than 6% of the volume of the colonies themselves (calculated from
data found in Sheridan et al. (2002) and assuming average diameters of 0.5, 10 and
10 µm of bacteria, diatoms and dinoflagellates, respectively). Thus, we believe that
the majority of the colony biomass and activity is Trichodesmium spp. itself and
therefore we assume that the kinetic and APA responses are primarily due to
Trichodesmium spp.
Sample collection: Colonies of Trichodesmium spp. were collected using either a 1
m, 202 μm mesh plankton net at a depth of 15-20 m or a 0.25 m, 64 μm plankton net
towed at the surface. Colonies were picked out of the tow with plastic inoculating
loops and placed in GF/F filtered seawater (FSW; nominal pore size 0.7 μm) to rinse.
33
P uptake: 5-10 rinsed colonies were placed in 50 ml of FSW in 60 ml
polycarbonate bottles with 0.5 – 6.25 µCi of H
3
33
PO
4
, added as 10-20 µl of a
working stock solution, amounting to a 2 – 20 fM addition of PO
4
3-
. All incubations
were run in triplicate. Samples were incubated in 50% of surface light in on-deck
incubators for 60-90 minutes, as time course experiments at the beginning of each
cruise indicated that
33
PO
4
3-
uptake was linear for about the first 90 minutes.
41
Experiments comparing light and dark PO
4
3-
uptake were performed by incubating
Trichodesmium spp. colonies in 50% surface light and in dark bottles in parallel. To
measure diel P uptake, samples were collected by net tow and incubated every 2-3
hours over the course of a day, beginning before sunrise and ending after sunset. To
generate kinetic curves, 0.025-0.5 µmol L
-1
K
2
HPO
4
was added to incubations. V
max
and K
s
were found by fitting the Michaelis-Menton equation to each data set with
regression analysis in Sigmaplot. The kinetics of nutrient uptake are described by
the equation V = (V
max
×S)/(K
s
+S), where V is the uptake rate at nutrient
concentration S (the SRP concentration measured in surface water plus the calculated
concentration of cold K
2
HPO
4
added – radiolabelled PO
4
3-
was added in
concentrations ~six orders of magnitude lower than measured SRP, and is thus not
included in this calculation), V
max
is the maximal, or saturated, uptake rate, and K
s
is
the nutrient concentration when uptake is one-half V
max
. SRP was always
measureable in surface waters (Atlantic and Pacific data were provided by Karin
Björkman (pers. comm.)).
Alkaline phosphatase activity: Alkaline phosphatase activity in Trichodesmium spp.
and bulk water was determined using the method described by (Ammerman 1993)
which uses an organic molecule, methylumbelliferone (MUF), with a PO
4
3-
attached
(MUF-P) as a substrate for alkaline phosphatase. Alkaline phosphatase cleaves the
PO
4
3-
moiety from MUF-P, causing it to fluoresce. Twenty-five to 40 rinsed
42
Trichodesmium spp. colonies were placed into 250 ml of unfiltered, bucket-collected
surface seawater with 100 nmol L
-1
(Atlantic) and 200 nmol L
-1
(Pacific) MUF-P
added. Samples were incubated in flowing seawater at 50% ambient light to
simulate in situ conditions. The activity of seawater alone was also measured, and
its activity subtracted from the activity of Trichodesmium spp. plus seawater to
obtain activity of Trichodesmium spp. alone. Bulk seawater activity was always
measureable and usually very low compared to Trichodesmium spp., but was
occasionally responsible for more than half the total activity in bottles with seawater
plus Trichodesmium colonies. Experiments were conducted in this manner because
previous experiments have shown that filtering seawater increases activity over
unfiltered water, possibly due to cell breakage and release of the phosphatase
enzyme.
The increase in fluorescence in the incubations was measured every hour for 4 to 7
hours by removing 3 ml of sample water and placing it into a glass test tube with 1
ml of 50 mmol L
-1
borate buffer, pH 10.8. The buffer raises the pH above 10, where
MUF becomes fluorescent. A Turner 10-AU fluorometer with a long wavelength oil
lab filter kit was used to read the fluorescence. The slope of fluorescence versus
time is compared to a standard of MUF, made up at the same concentration as the
substrate added. We report the turnover rate (h
-1
) of MUF-P by calculating t = slope
of fluorescence vs. time/fluorescence of the standard, then normalizing values to the
43
biomass parameters chl a content or trichome number. This corrects for the effect
that higher substrate concentrations in the Pacific would have on the hydrolysis rate.
RESULTS
Light vs. dark and diel P uptake: Unlike CO
2
and N
2
fixation in Trichodesmium
spp., PO
4
3-
uptake is not dependent on light or time of day. On any given day when
light and dark PO
4
3-
uptake were measured in the Atlantic, either of the treatments
may have had higher uptake rates than the other (Figure 2-2).
light PO
4
uptake (pmol col
-1
h
-1
)
02 4 6 8 10 12
dark PO
4
uptake (pmol col
-1
h
-1
)
0
2
4
6
8
10
12
Figure 2-2 PO
4
3-
uptake in 50% of surface light versus the dark for colonies
collected in the north Atlantic. The 1:1 line is shown on the graph.
44
A paired t test of the entire data set shows there was no significant difference
between the two (p > 0.2). When PO
4
3-
uptake was measured over a diel cycle (3
one day cycles determined), ambient uptake varied over the course of the day but no
consistent pattern was observed (Figure 2-3).
Time of day
05:00 09:00 13:00 17:00
PO
4
uptake (pmol P col
-1
h
-1
)
0
1
2
3
4
5
6
Atlantic 15May03
Pacific 11Aug03
Pacific 19Aug03
Figure 2-3 Per colony PO
4
3-
uptake rate measured throughout the day (sunrise to sunset) in
the north Atlantic and Pacific. Error bars show standard error of three replicates.
P uptake kinetics: Kinetic parameters of PO
4
3-
uptake in Trichodesmium spp. were
found for experiments where uptake was saturated or near saturated. There is fairly
high variability in the data within the sample locations (Table 2-1), however,
differences can be seen among locations. V
max
in the tropical North Atlantic
45
averaged 6.5 nmol P μg chl a
-1
hr
-1
, 5.4 and 3.8 times greater than in the subtropical
North Pacific and off the north coast of Australia, respectively (Table 2-2 and Figure
2-4).
Table 2-1 V
max
and APA at station in the North Atlantic, North Pacific and north of Australia. Values
are shown normalized to both chl a and number of trichomes per colony
Date V
max
(nmol P μg chl a
-1
h
-1
)
APA
(h
-1
μg chl a
-1
)
V
max
(pmol P trich
-1
h
-1
)
APA
(h
-1
trich
-1
×10
-6
)
Atlantic
22Apr03 2.6 - 0.25 -
24Apr03 4.5 - 0.068 -
25Apr03 4.0 - 0.16 -
26Apr03 - 0.70 - 38
27Apr03 6.0 0.052 0.57 4.9
30Apr03 13.8 - 1.1 -
01May03 10.8 0.045 0.55 2.3
03May03 - 0.074 - 6.0
10May03 6.1 - 0.69 -
11May03 3.9 - 0.54 -
12May03 - 0.10 - 4.1
13May03 - 0.52 - 5.2
15May03 - 0.090 - 7.9
Pacific
10Aug03 0.64 0.083 0.055 7.1
10Aug03 0.93 - 0.079 -
12Aug03 0.33 0.012 0.095 3.6
14Aug03 - 0.017 - 1.1
15Aug03 2.8 0.037 0.32 2.9
17Aug03 2.6 0.0054 0.21 0.69
18Aug03 0.18 0.062 0.023 7.2
Australia
18Nov99 4.0 - 0.14 -
19Nov99 4.0 - 0.19 -
20Nov99 0.76 - 0.056 -
21Nov99 0.22 - 0.018 -
24Nov99 0.44 - 0.030 -
26Nov99 0.73 - 0.028 -
46
Table 2-2 Summary of PO
3-
4
uptake kinetics and APA, surface nutrient concentrations, and
Trichodesmium spp. colony information in the North Atlantic, North Pacific and north of Australia
shown with standard error in parentheses.
North Atlantic North Pacific Australia
V
max
(nmol P µg chl a
-1
h
-1
) 6.5 (1.4)
1
1.2 (0.5) 1.7 (0.7)
V
max
(pmol P trichome
-1
h
-1
) 0.50 (0.12)
2
0.13 (0.05) 0.078 (0.029)
K
s
(µM) 0.18 (0.04) 0.15 (0.04) 0.15 (0.05)
APA (µg chl a
-1
h
-1
) 0.16 (0.09) 0.036 (0.013) -
APA ( ×10
-6
trichome
-1
h
-1
) 9.7 (4.7) 3.8 (1.2) -
SRP (µM)
3
0.045 (0.005)
4
0.036 (0.006)
5
0.11 (0.02)
dFe (nM)
6
2.4 (0.4)
7
0.35 (0.06) 0.37 (0.06)
SRP:dFe 18.8 103 297
trichomes per col
8
149 (26) 103 (19)
9
204 (7)
chl a per col
8
0.0095 (0.0014) 0.013 (0.004) 0.012 (0.002)
N
2
fixation (nmol N col
-1
d
-1
)
10
0.093 0.12 0.15
1
Significantly different from V
max
in the Pacific (p=0.008) and Australia (p=0.016)
2
Significantly different from V
max
in the Pacific (p=0.029) and Australia (p=0.013)
3
Data from Karin Björkman (Atlantic and Pacific). Australia data from Doug Capone (unpubl.)
4
Significantly different from SRP in the Pacific (p=0.016) and Australia (p=0.002)
5
Significantly different from SRP north of Australia (p=0.002)
6
dFe defined as the [Fe] in the <0.2 µm filtrate. Data from Sergio Sanudo-Wilhelmy (Atlantic,
personal communication), Brown et al. (2005; Pacific, from 160-150ºW and 22-28ºN) and Adam
Kustka (Australia, personal communication)
7
Significantly different from dFe in the Pacific (p=0.003) and Australia (p=0.003)
8
Data from Ed Carpenter (personal communication)
9
Significantly different from trichomes per colony north of Australia (p=0.002)
10
At 55% light level. Atlantic data from Capone et al. (2005). Pacific data from Doug Capone
(unpubl)
47
PO
4
uptake (pmol P trichome
-1
h
-1
)
0.00
0.02
0.04
0.06
0.08
0.10
PO
4
uptake (pmol P trichome
-1
h
-1
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
PO
4
concentration ( μM)
0.00 0.05 0.10 0.15 0.20 0.25
PO
4
uptake (pmol P trichome
-1
h
-1
)
0.000
0.005
0.010
0.015
0.020
Figure 2-4 Examples of PO
4
3-
uptake kinetic curves from the North Atlantic (top panel),
North Pacific (middle panel) and waters north of Australia (bottom panel). Note that the
scales on the Y axes are different. Error bars show standard error of three replicates.
48
A two-tailed t test showed that V
max
in the North Atlantic was significantly different
from both the North Pacific (p = 0.008) and north of Australia (p = 0.016), but there
was no significant difference between the North Pacific and North of Australia (p =
0.63). When normalized to number of trichomes per colony, V
max
is still statistically
significantly different when comparing the North Atlantic to the North Pacific (3.8
fold greater, p = 0.029), or to waters north of Australia (6.5 fold greater, p = 0.013)
(Table 2-2).
While differences were apparent in V
max
, the half saturation constant was not
different among the three locations. K
s
averaged 0.18 μmol L
-1
in the Atlantic, 0.15
μmol L
-1
in the Pacific and 0.15 μmol L
-1
north of Australia (Table 2-2). K
s
was not
statistically significantly different for any of the comparisons (p>0.6 for all
comparisons).
Alkaline phosphatase activity: Alkaline phosphatase activity was also quite variable
at each location (Table 2-1), but was higher in the North Atlantic than the North
Pacific, on average. The turnover rate of MUF-P was 0.16 μg chl a
-1
hr
-1
in the
North Atlantic, 4.4 times greater than in the North Pacific. APA was 2.6 times
greater in the North Atlantic than the North Pacific when normalizing to number of
trichomes per colony (Table 2-2). However, neither of these comparisons showed a
significant difference (p>0.2).
49
DISCUSSION
Light vs. dark and diel P uptake: CO
2
and N
2
fixation are both reductive processes
and therefore energetically demanding. Both processes are light dependent in
Trichodesmium spp. and show diel patterns that are externally forced (Berman-Frank
et al. 2001; Capone et al. 1990; Zehr et al. 1993; Capone et al. 2005), and both
processes are dependent on energy generated by photosynthesis. This does not
appear to be true for uptake of PO
4
3-
, which does not undergo redox transformation
during uptake and assimilation. In natural populations of Trichodesmium spp.,
uptake of PO
4
3-
is neither dependent on the presence of light nor time of day. PO
4
3-
is
found at very low levels in the oligotrophic environments where Trichodesmium spp.
occurs. Hence, an adaptive strategy would be to assimilate PO
4
3-
whenever and
wherever it is encountered.
Studies with cultured cyanobacterial species and field populations have yielded
similar results to those reported here. Fu et al. (2005) showed that PO
4
3-
uptake in 2
different Trichodesmium erythraeum strains grown at the same light level was the
same when placed in light or dark. Additionally, neither Phormidium laminosum
(Prieto et al. 1997), a freshwater cyanobacterium, nor Synechococcus WH7803
(Donald et al. 1997), a marine cyanobacterium, showed a difference in PO
4
3-
uptake
in the light or dark. PO
4
3-
uptake in the Northeast Atlantic was generally the same in
50
the light and the dark (Donald et al. 2001) and PO
4
3-
uptake in samples from the
North Pacific Subtropical Gyre (NPSG) showed no diel rhythm (Björkman et al.
2000).
P uptake kinetics: Kinetic parameters of PO
4
3-
uptake varied widely both within and
among sites. However, V
max
was four times greater or more in the North Atlantic
than in the North Pacific or north of Australia when normalized to either chl a
content and trichome number. Increased V
max
is a common response in
phytoplankton to P limitation. Fu et al. (2005) reported that V
max
of PO
4
3-
uptake in
a culture of Trichodesmium (erythraeum) IMS 101 was 0.68 pmol P trichome
-1
h
-1
in
P deplete conditions (assuming 50 cells trichome
-1
) and 0.12 pmol P trichome
-1
h
-1
when P replete, strikingly similar to our values of 0.50 pmol P trichome
-1
h
-1
in the
North Atlantic and 0.13 and 0.078 pmol P trichome
-1
h
-1
in the North Pacific and
north of Australia, respectively (Table 2-2). Other species of laboratory cultures
show similar results. V
max
can increase by an order of magnitude or more in cultures
of diatoms (Perry 1976; Donald et al. 1997) and cyanobacteria (Donald et al. 1997;
Ikeya et al. 1997). These results strongly support the conclusion that field
populations of Trichodesmium spp. are P stressed in the North Atlantic, but not in the
North Pacific or along the north coast of Australia.
51
It is important to note that there can be differences between Trichodesmium spp.
isolates in V
max
of PO
4
3-
uptake, but that these are largely a function of size
differences (Fu et al. 2005). The predominant species we encountered north of
Australia was T. erythraeum, while T. thiebautii dominated in both the North
Atlantic and Pacific. The typical diameter of a trichome of T. erythraeum is 20 µm,
while T. thiebautii are more variable and can range from 6-20 µm (Siddiqui et al.
1992). Therefore, the trichomes from the north of Australia were likely larger than
trichomes from the other two locations. Despite their size, trichome normalized
values in waters north of Australia were toward the lower end of values observed
among the three sites, which would suggest that Trichodesmium spp. from these
waters may be even less stressed than the data suggest. Trichodesmium spp.
collected from the North Pacific and North Atlantic were dominated by the same
species and so are more directly comparable. Thus, the species differences between
Trichodesmium spp. from Australia and the North Atlantic and Pacific should not
affect the overall conclusions of our research.
Another common kinetic response to P stress is a decrease in the half saturation
constant caused by the induction of a second, higher affinity PO
4
3-
transport system
(Donald et al. 1997; Dyhrman and Haley 2006). However, not all species show this
response. K
s
decreased by an order of magnitude in two species of Synechococcus
grown under P limitation (Donald et al. 1997; Ikeya et al. 1997), but was nearly the
52
same in cultures of T. weisflogii and E. huxleyi grown in P deplete and replete
conditions (Donald et al. 1997; Riegman et al. 2000). Trichodesmium erythraeum
cultures showed no difference in K
s
under P replete and deplete conditions (Fu et al.
2005). K
s
was not statistically significantly different among the three locations in
our study either – locations that showed differential P stress in Trichodesmium spp..
This suggests that Trichodesmium spp. does not possess a second, high affinity,
PO
4
3-
transporter that is induced by P limitation.
Alkaline phosphatase activity: Alkaline phosphatase activity is a commonly used
assay to test for P stress. The results from this study indicate that Trichodesmium
colonies in the North Atlantic are more P stressed than colonies found in the North
Pacific. When combining this data with the maximal uptake rates found on the same
cruises, the argument for relatively greater P limitation of Trichodesmium spp. in the
North Atlantic compared to the North Pacific or north of Australia, is even stronger.
However, it does appear that V
max
is a more sensitive indicator of P stress, as the
difference in V
max
between the North Atlantic and North Pacific was greater than the
difference in APA. Other studies of APA in the Atlantic also suggest P stress there.
Large areas of the Atlantic show P stress, as measured with APA, in bulk water
samples (Vidal et al. 2003) and Trichodesmium spp. APA measured in the western
tropical Atlantic near the Caribbean Sea is extremely high compared to Australia (2
53
orders of magnitude; Mulholland et al. 2002), indicating Trichodesmium spp. in the
Caribbean Sea may be even more stressed for P than the areas in this study.
Nutrient limitation – comparison to other studies: The results of this study indicate
that P is the major nutrient control on Trichodesmium spp. in the North Atlantic, but
not in the North Pacific or north of Australia. This agrees with other published
assessments of open ocean nutrient limitation. Krauk et al. (2006) used particulate N
to P ratios of Trichodesmium spp. as a relative measure of P stress: higher N:P ratios
in culture were related to SRP depletion. In the field, N:P ratios averaged ~50 in the
western North Atlantic, but 40 in the North Pacific and 22 north of Australia,
showing that western North Atlantic Trichodesmium spp. was relatively more P
stressed. (The North Pacific and Australia data were collected on the same cruises as
in this study.) Trichodesmium spp. P quotas in the North Atlantic also positively
correlate to N
2
fixation rates (Sañudo-Wilhelmy et al. 2001). Additionally, enzyme
labeled fluorescence of Trichodesmium spp. alkaline phosphatase shows that AP is
active in the North Atlantic (Dyhrman et al. 2002) but almost non-existent in the
North Pacific (Hynes et al. Pers. Comm.).
Other studies also support the idea of P limitation in the North Atlantic. Water
column nutrient data (Wu et al. 2000) and modeling efforts (Moore et al. 2002) both
suggest that N
2
fixers should be P limited in the North Atlantic and Fe limited in the
54
North Pacific. However, declining SRP stocks in the North Pacific over the past
decades (Karl and Tien 1997) along with a near doubling of Trichodesmium
filaments in the period from 1989-1992 (Letleier and Karl 1996) led researchers to
suggest that these organisms were depleting P and driving the system to P limitation
(Karl et al. 1997). Our results do not support this hypothesis for Trichodesmium
spp., a dominant N
2
fixer. Measures of P stress are low in the NPSG compared to
the North Atlantic. Moreover, our results were obtained during July and August
when limitation might also be expected to be maximal due to seasonally low SRP
inventories driven by increased production in the summer months (Karl et al. 1996).
Our results on the apparent lack of P stress of Trichodesmium spp. in the North
Pacific suggest that another factor or factors limit diazotrophs in the NPSG.
Finally, we can speculate on the mechanisms leading to observed patterns in these
three systems. Presumably, Trichodesmium spp. found north of Australia is not P
limited because measurable and often high concentrations (0.27 μmol L
-1
at one site)
of SRP
were found there, perhaps due to the generally shallow depths (< 100 m) and
proximity to the coastline of many of the stations on this cruise. Experimental Fe
additions to colonies collected north of Australia suggest Fe is limiting to
Trichodesmium spp. in this location at least some of the time (Kustka et al. 2003a).
Dust deposition, a major source of Fe to remote regions, is much greater in the N.
Atlantic than the N. Pacific and north of Australia (Gao et al. 2001). Indeed, Fe
55
concentrations in surface waters reflect this difference. Dissolved Fe concentrations
measured on the Australia cruise were similar to reported values in the North Pacific
near Hawaii and much lower than dissolved Fe concentrations on our North Atlantic
cruise (Table 2-2). Additionally, Fe deposition to the North Atlantic through aeolian
dust is estimated to be double that to the North Pacific and waters north of Australia
(Gao et al. 2001). In contrast, SRP concentrations in the North Pacific are
statistically the same as those found in the North Atlantic, yet V
max
and APA are
much higher in the North Atlantic. When examining the SRP:dFe ratios for the three
locations, surface waters of the North Atlantic have the lowest ratio by far (Table 2-
2); these waters are enriched in Fe compared to P. Similar to the hypothesis
proposed by Wu et al. (2000), we posit that the abundance of Fe in the North
Atlantic drives Trichodesmium spp. to P limitation there, while the paucity of Fe in
the North Pacific and north of Australia renders P limitation less important there.
Interestingly, colony specific rates of N
2
fixation were similar among the three
locations (Table 2-2), suggesting that limitation by different nutrients does not
necessarily affect fixation rates differently. However, the response to changes in
nutrient delivery – an increase in dust deposition or P weathering for example –
would be very different among the different locations. Therefore, the differential
nutrient limitation of Trichodesmium spp. in the areas where it occurs (e.g.
hypothesized Fe limitation in the North Pacific), and the response to limiting nutrient
inputs, is a topic requiring further study.
56
The globally significant diazotroph Trichodesmium spp. responds to P stress by
altering its ability to acquire P from both organic and inorganic sources. Maximal
uptake of PO
4
3-
increases greatly under P limitation of Trichodesmium spp., as does
APA. However, laboratory data (Fu et al. 2005) along with the data collected in this
study, show that the half saturation constant (K
s
) is not reduced in Trichodesmium
spp., as occurs in many other phytoplankton species, and therefore does not appear to
induce a secondary, high affinity, PO
4
3-
transporter under P stress. Thus, V
max
and
APA can thus be used as a diagnostic indicator of P limitation or stress, while K
s
can
not. Data collected in the tropical North Atlantic, subtropical North Pacific and off
the northern coast of Australia show that Trichodesmium spp. in the Atlantic is P
limited relative to the Pacific and north of Australia. Other studies suggest that P is
limiting in the Atlantic because of the abundance of Fe delivered in Saharan dust (or,
alternatively, from rivers; Tovar-Sanchez et al. 2006) and is not limiting in the
Pacific because Fe is in such short supply (Wu et al. 2001, Gao et al. 2001).
Australia is likely not P limited because SRP is about 2.5 times greater than in the
Atlantic and Pacific sites. Our data provide direct diagnostic evidence that there are
different primary limiting factors for Trichodesmium spp. in the tropics of the North
Atlantic compared to the North Pacific, and underscore the need to delineate the
divergent nutrient dynamics that lead to this condition.
57
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in the central Atlantic Ocean indicate large areas with phosphorus deficiency. Marine
Ecology-Progress Series 262: 43-53.
Wu, J., W. Sunda, E. A. Boyle, and D. M. Karl. 2000. Phosphate depletion in the western North
Atlantic Ocean. Science 289: 759-762.
Wu, J., E. Boyle, W. Sunda, and L. Wen. 2001. Soluble and colloidal iron in the oligotrophic North
Atlantic and North Pacific. Science 293: 847-849.
Zehr, J. P., M. Wyman, V. Miller, L. Duguay, and D. G. Capone. 1993. Modification of the Fe protein
of nitrogenase in natural populations of Trichodesmium thiebautii. Applied and
Environmental Microbiology 59: 669-676.
61
CHAPTER III: Phosphorus dynamics of the tropical and subtropical north Atlantic:
Trichodesmium vs. bulk plankton
CHAPTER III ABSTRACT
Dinitrogen (N
2
) fixing organisms such as Trichodesmium spp. are abundant in the
oligotrophic tropical North Atlantic Ocean, where microplankton (including other
diazotrophs) are more likely to be phosphorus (P) than nitrogen (N) limited. Thus,
understanding ability of different functional groups in the plankton to compete for P
in this area is important in understanding their relative success. The uptake of
phosphate by Trichodesmium spp. colonies and bulk water plankton was measured
using
33
PO
4
-3
over a range of concentrations, and kinetic parameters were
determined. Nano- and pico-plankton present in bulk water samples have a K
s
nearly
30 times lower than that of Trichodesmium spp. While chlorophyll-a normalized
alkaline phosphatase activity (APA) in bulk water was an order of magnitude greater
than in Trichodesmium spp., Trichodesmium spp. contributes substantially to total
APA in the water. Trichodesmium spp. is outcompeted for inorganic P, but colonies
can satisfy their P needs by supplementing DIP uptake with P cleaved from DOP via
alkaline phosphatase.
62
INTRODUCTION
Phosphorus (P) has a very long residence time in the oceans (Codispoti 1989),
although estimates have been recently revised downwards due to new analyses
(Benitez-Nelson 2000). The cycling of this macronutrient has long been viewed as
primarily controlled by chemical processes occurring on geological time scales, with
the conversions that happen in the biological realm being incidental (Tyrell 1999,
Benitez-Nelson 2000). This stems from the common view that the biological
productivity of the world’s oceans is primarily nitrogen (N) limited, with changes in
P cycling (Benitez-Nelson 2000) or N inventories being tempered through the
regulation of dinitrogen (N
2
) fixation and denitrification (Michaels et al. 2001).
However, certain areas of the globe are now thought to be P limited (e.g. the
Mediterranean (Thingstad et al. 2005)), and thus the study of P dynamics in these
areas is essential.
Trichodesmium spp. is a N
2
fixing, colony forming cyanobacteria that occurs in
many areas of the ocean where warm, calm waters and oligotrophic conditions
prevail (Capone et al. 1997). Colonies can take on either the tuft formation (parallel
alignment of trichomes) or the puff formation (radially arranged trichomes). The
diazotrophic nature of Trichodesmium spp. means that it is never limited by N, and
thus other nutrients are important in regulating their growth and N
2
fixation. Both P
63
and Fe have been suggested as limiting to diazotrophs, but it appears most likely that
different nutrients may limit N
2
fixers in different areas of the ocean. High
concentrations of Fe from the large fluxes of dust from the African continent (Wu et
al. 2000) and high densities of N
2
fixing organisms such as Trichodesmium spp.
(Carpenter et al. 2004, Capone et al. 2005) create conditions conducive to P
limitation in the tropical and subtropical North Atlantic (Wu et al. 2000, Sañudo-
Wilhelmy et al. 2001). Studies show that both the bulk water plankton (made up in
large part by heterotrophic bacteria and the cyanobacteria Prochlorococcus and
Synechococcus (Li et al. 1992)) and Trichodesmium spp. are P limited in the North
Atlantic (Cotner et al. 1997, Sañudo-Wilhelmy et al. 2001, Ammerman et al. 2003,
Krauk et al. 2006). The apparent limitation by P of these various groups means that
they must compete with each other for P from both the inorganic and organic pools.
Both picoplankton and Trichodesmium spp. can access the inorganic and organic
pools of P through P transport enzymes and alkaline phosphatase. In addition,
BLAST searches have found sequences in the genome of Trichodesmium erythraeum
IMS 101 for phosphonate transport and metabolism proteins, indicating
Trichodesmium spp. may be able to exploit this often overlooked component of DOP
(genome.ornl.gov/microbiology/tery, Dyhrman et al. 2006).
How, exactly, a Trichodesmium colony is able to acquire enough P to fulfill its
cellular requirements when it grows in areas with such low PO
4
-3
concentrations has
64
been an issue of some debate for years, with some suggesting that colonies might
migrate towards the phosphocline when P-deplete to take up large amounts of P and
then return to the surface (Karl et al. 1992). However, if Trichodesmium spp. can
successfully compete for either DIP or DOP, it may be able to acquire the necessary
P for growth without migration to the phosphocline.
To assess the ability of Trichodesmium spp. to compete with bulk plankton for P in a
P limited system, we measured PO
4
-3
uptake kinetics and alkaline phosphatase
activity for both samples types in the subtropical and tropical western north Atlantic
in March of 2004.
MATERIALS AND METHODS
Sample collection: Experiments were performed aboard the R/V Endeavor in March
of 2004 on a southeast transect from about 30 °N, 65 °W down to 10°N, 50 °W (Figure
3-1). Samples were collected by towing a 0.25 m, 202 μm mesh net at a depth of 15-
20 m for about 10 minutes at a time. Colonies were found in both the puff and tuft
formations, with puffs outnumbering tufts. Colonies were picked out of the tow
sample using a plastic inoculating loop and placed into filtered seawater to rinse.
The colonies were then picked out of the rinse and placed in the bottles used for the
different assays. Efforts were made to place the different colony conformations into
65
assays at approximately the same abundance they were found in the tow. Colonies
were not obtained at all stations, particularly those at the northern end of the transect.
Bulk plankton was collected from either a clean underway seawater system or from
near the surface (< 2 m depth) sampled by a Niskin bottle on a CTD rosette system.
Figure 3-1 A map of the area studied in the subtropical
and tropical North Atlantic showing station locations.
66
PO
4
-3
uptake: Fifty ml samples of bulk seawater or filtered seawater containing 10
Trichodesmium colonies were placed in 60 ml acid washed polycarbonate bottles
with 0.5 – 2 µCi of
33
PO
4
-3
(final concentration 2 – 8 fM PO
4
-3
) and incubated in
25% light on deck incubators for 60-90 minutes. Incubations were filtered onto
polycarbonate filters, 8 µm for Trichodesmium colonies and 0.2 µm for bulk
seawater samples. Incubation bottles were rinsed three times with 0.2 µm filtered
seawater, the rinse poured over the filters, with one final filtered seawater rinse of
the filter towers before the filters were placed into 7 ml plastic scintillation vials.
Activity of
33
P was measured on board in a scintillation counter after addition of 5 ml
of scintillation cocktail. Experiments were conducted on this cruise and on previous
cruises that showed DIP uptake in Trichodesmium spp. is linear for the first 60-90
minutes. Other studies have shown that DIP uptake in bulk samples of seawater is
linear for many hours (Björkman et al. 2000).
The Michaelis-Menten equation is used to describe the uptake kinetics of nutrients.
By fitting the equation: V = V
max
*S/(K
s
+S), to a plot of PO
4
-3
concentration in an
incubation (measured DIP plus calculated cold PO
4
-3
added) versus the PO
4
-3
uptake
rate at that concentration, one can solve for the rate of maximal uptake (V
max
) and the
half saturation constant (K
s
). To create kinetic curves, 0 to 1 µM of cold PO
4
-3
was
added to incubations, followed immediately by radiolabeled PO
4
-3
addition. Samples
were then treated as described above. SigmaPlot was used to directly fit the
67
Michaelis-Menten equation to the data and extract V
max
and K
s
(K
s
was corrected for
the amount of SRP measured in surface water). It has been recently shown that a
significant amount of P in a colony is sorbed to the outside of the cells, and washing
sorbed P from the outside of the cells is important in measuring actual P uptake with
33
PO
4
-3
(Sañudo-Wilhelmy et al. 2004). To control for this abiological adsorption of
P, a killed control was always conducted with addition of 1 ml of glutaraldehyde to
measure abiological adsorption to colonies and the calculations for P uptake were
corrected for this. Uptake of the radioisotope in killed controls of Trichodesmium
spp. was 8% (on average) of the uptake of
33
P into live colonies.
Alkaline phosphatase activity: The PO
4
-3
moiety is cleaved from DOP at the cell
surface and taken up as inorganic PO
4
-3
, and thus will not be accounted for in
measurements of PO
4
-3
uptake with respect to ambient PO
4
-3
concentration. It is
therefore important to measure the activity of alkaline phosphatase separate from
PO
4
-3
uptake to assess the potential for uptake of phosphate from the DOP pool.
The method described by Ammerman (1993) was used to measure APA. Briefly,
250 ml samples of surface seawater or 30 colonies of Trichodesmium spp. in
unfiltered surface seawater were incubated with 100 nM methyllumberiferone-
phoshpate (MUF-P). Alkaline phosphatase cleaves the PO
4
-3
moiety from the
molecule, causing it to fluoresce. Samples were incubated in on-deck incubators at
68
25% light and the increase in fluorescence of MUF was measured over the course of
the day, usually over a 6 – 7 hour period, using a Turner 10-AU fluorometer with a
long wavelength oil lab filter kit. APA was calculated using the linear portion of the
slope of MUF fluorescence versus time, relative to a 100 nM MUF standard, and the
concentration of DOP in the water. For assays on Trichodesmium spp., the rate of
APA measured in seawater was subtracted from the activity of Trichodesmium spp.
plus surface seawater to obtain activity of Trichodesmium spp. alone. Experiments
were conducted in this manner because in previous experiments we have seen that
filtering seawater can increase activity over unfiltered water, possibly due to cell
breakage and release of the phosphatase enzyme.
Chlorophyll a and Nutrient analyses: Chlorophyll concentrations were determined
by filtering 1 liter of seawater onto a GF/F glass fiber filter, extracting the
chlorophyll with acetone for 24 hours, then reading the sample on a fluorometer set
to detect chl a and comparing it to a known standard (Holm-Hansen and Riemann
1978). Chlorophyll from colonies of Trichodesmium spp. was also extracted in
acetone and measured on a fluorometer after 24 hours. Surface samples for DIP
concentration were collected in acid washed plastic vials and measured with the
MAGIC method (Karl and Tien 1992). Samples were concentrated by a factor of
six, making the limit of detection 5 nM. Total dissolved phosphorus (TDP) was
measured on unfiltered samples (as PP was assumed to be <10% of total P) using the
69
high temperature combustion and acid hydrolysis method of (Solorzano & Sharp
1980). Dissolved organic phosphorus (DOP) was calculated by subtracting DIP
from TDP.
RESULTS
Inorganic and organic P concentrations in this area of the North Atlantic Ocean were
found to be very low at this time of year, with DIP averaging 0.04 µM, while DOP
concentrations were almost three times greater at 0.11 µM (Table 3-1). Chlorophyll
a showed about a 10-fold range of concentrations along the transect: from 0.018 –
0.22 µg l
-1
, while Trichodesmium spp. chlorophyll content was 8 – 27 ng col
-1
.
Chlorophyll normalized APA in bulk water was similar at most stations. However,
station 3 showed an extreme rate of 83.9 nmol P µg chl a
-1
h
-1
,
much higher than at
the other stations. On average, bulk water APA was 19.4 ± 32.1 nmol P µg chl a
-1
h
-
1
. APA for Trichodesmium spp. was measured at three stations, and the activity
measured averaged 5.9 ± 3.8 nmol P µg chl a
-1
h
-1
. When comparing average
Trichodesmium spp. APA to bulk water APA, chl specific bulk water APA was four
times greater than Trichodesmium spp. over the entire region studied. However, if
the high value from station 3 is removed, they are nearly the same.
70
Table 3-1 Concentrations of chl a, inorganic and organic phosphorus in the North Atlantic Ocean and inorganic P uptake and alkaline
phosphatase activity of bulk water samples and Trichodesmium colonies at ambient DIP and DOP concentrations. At station 6, bulk
water and Trichodesmium PO
4
3-
uptake rates were determined twice.
Station DIP
(µM)
DOP
(µM)
Bulk
chl a
(µg l
-1
)
Tricho
chl a
(µg col
-1
)
Bulk APA
(nmol P µg chl a
-1
h
-1
)
Tricho APA
(nmol P µg chl a
-1
h
-1
)
Bulk PO
4
uptake
(nmolP µg chl a
-1
h
-1
)
Tricho PO
4
uptake
(nmolP µg chl a
-1
h
-1
)
2 0.05 0.17 0.12 17.6 25.0
A 0.01 0.18 0.22 1.5 2.8
B 0.02 0.09 0.21 7.1
3 0.02 0.018 0.015 83.9 61.1 0.02
C 0.01 0.06 0.026 3.9 53.8
D 0.01 0.07 0.035 48.6
4 0.08 0.027 4.5 0.51
E 0.04 0.10 0.051 6.5 154.9
F 0.03 0.12 0.039 74.4
5 0.06 0.019 3.1 0.27
6 0.07 0.128 0.008/0.008 3.1 10.3 69.5/43.8 1.03/0.62
Average 0.04 0.11 0.16 0.015 19.4 5.9 54.1 0.53
71
The chl normalized uptake of PO
4
3-
at ambient concentrations in bulk water samples
measured with
33
P was nearly a factor of three greater than uptake from the organic
pool (as measured from APA), while inorganic P uptake in Trichodesmium spp. was
an order of magnitude less than APA.
Phosphate uptake kinetic curves were determined for bulk water at 2 stations (B and
C) and Trichodesmium spp. at 2 stations (5 and 6), with 2 curves produced at station
6. An example of each can be seen in Figure 3-2.
V
max
averaged 12.2 ± 2.1 nmol P µg chl a
-1
h
-1
for bulk samples and 10.3 ± 4.7 nmol
P µg chl a
-1
h
-1
for Trichodesmium spp. (Table 3-2). K
s
for bulk plankton was 0.015
µM for both of the stations where it was measured, while K
s
for Trichodesmium spp.
was 0.78 ± 0.37 µM on average, 50 times greater than for bulk plankton (Table 3-2).
The affinity of bulk plankton and Trichodesmium spp. for PO
4
3-
can be calculated as
V
max
/K
s
(also known as α), and was 0.81 and 0.016 L μg chl a
-1
h
-1
, respectively.
72
PO
4
concentration ( μM)
0.0 0.2 0.4 0.6 0.8 1.0 1.2
PO
4
uptake (nmol P* μg chl a
-1
*hr
-1
)
0
2
4
6
8
10
0.00 0.05 0.10 0.15 0.20
PO
4
uptake (nmol P μg chl a
-1
h
-1
)
0
2
4
6
8
10
12
14
a
b
Figure 3-2 Examples of kinetic curves for bulk water samples (a) and Trichodesmium colonies (b).
Panel A is from station B while panel b is from station 6. PO
4
-3
concentration in these graphs
represents amount of cold PO
4
-3
added to incubations plus ambient PO
4
-3
concentration in surface
water. Note that the scale on the X and Y axes are different for each graph
73
Station V
max
(nmol P µg chl a
-1
h
-1
)
K
s
(µM)
α
(l μg chl a
-1
h
-1
)
Bulk
B 13.6 0.015 0.91
C 10.7 0.015 0.71
Trichodesmium
5 5.5 0.85 0.006
6 10.8 0.38 0.028
6 14.8 1.1 0.013
Table 3-2 V
max
, K
s
, and α for bulk water and Trichodesmium
samples. Parameters were found by fitting the Michaelis-Menten
equation to the data collected. Alpha, the affinity constant, and is
equal to V
max
/K
s
DISCUSSION
Based on previous studies (Cotner et al. 1997, Rivkin & Anderson 1997,
Obernosterer et al. 2003) and rapid PO
4
3-
pool turnover times in the surface of about
10 hours measured on this cruise (data not shown), the area in the western Atlantic
from 30 °N to 10 °N appears to be strongly P limited. The relatively high surface
concentrations of Fe (Wu et al. 2001), comparisons of N
2
fixation rates with colony
P content (Sañudo-Wilhelmy et al. 2001) and particulate N:P ratios that are very high
(Krauk et al. 2006), have led others to conclude that P exerts a major control on
Trichodesmium spp. growth specifically, and diazotroph growth in general, in the
tropical and subtropical North Atlantic. Dyhrman et al. (2002) also concluded that
Trichodesmium spp. colonies in this region are severely P stressed using an ELF
74
assay which visualizes P stressed trichomes. Our results on Trichodesmium spp.
populations in this area also point to P limitation. V
max
values are eleven and seven
times greater than those found in the North Pacific Subtropical Gyre and off the
north coast of Australia (Sohm et al. in prep), areas that may be less P limited than
the North Atlantic. Thus, competition for P among the planktonic organisms in the
North Atlantic may be a defining feature of this ecosystem.
Chlorophyll a normalized uptake rates of inorganic P and alkaline phosphatase
activity were both higher in bulk plankton than in Trichodesmium spp., as was the
calculated V
max
. It is important to recognize that a portion of the activities measured
in bulk samples are carried out by heterotrophic bacteria, and thus these organisms
are not represented in the chlorophyll measurements. This problem can be overcome
by estimating volumetric rates (Table 3-3), and it can be seen that the patterns still
hold.
DIP uptake
(nM h
-1
)
APA
(nM h
-1
)
15% APA
a
(nM h
-1
)
% of total
DIP uptake
% of total
APA
Bulk plankton 4.8 ± 4.2 0.65 ± 0.74 0.10 99.9 89.5
Trichodesmium 0.0067 ± 0.0042 0.076 ± 0.028 0.011 0.1 10.5
Table 3-3 Average volumetric rates of inorganic P uptake and alkaline phosphatase
activity for bulk plankton and Trichodesmium, assuming 1 colony per liter of water
(Carpenter et al. 2004), and the percentage of total uptake or activity each group is
responsible for.
a
Column shows APA if only 15% of DOP is bioavailable, the upper limit found by
Björkman and Karl (2003)
75
While there are measurements of P uptake and APA in different areas of the Atlantic
for either bulk water plankton or Trichodesmium spp., ours is the first study to
directly compare these enzyme activities in both bulk samples of water and
Trichodesmium spp.. Donald et al. (2001) measured P uptake by bulk plankton along
a transect at 20ºW between 57.5ºN and 37ºN and found rates of 0.42 to 1.7 nM h
-1
,
which are comparable to, but less than the average volumetric P uptake found in our
study of 4.8 nM*h
-1
(Table 3). Maximal PO
4
3-
uptake found at stations in the
Sargasso Sea near Bermuda was 0.78 nM h
-1
in August and 0.82 nM h
-1
in March
(Cotner et al. 1997), less than ambient uptake rates found for this study. However, P
uptake in our study was generally lower at the more northern stations where Cotner
et al. (1997) collected their data, and comparable to the rates found in their study.
Maximal alkaline phosphatase activity found in the same study was 1.39 nM h
-1
in
August and 2.7 nM h
-1
in March. While this is higher on average than the rates we
found (0.65 nM h
-1
) the APA found at the Bermuda Atlantic Time Series station,
near where the Cotner data was collected, was 2.13 nM h
-1
, which is in line with the
Cotner et al. (1997) study.
Trichodesmium spp. APA found in the present study is within the range of activities
found for Trichodesmium spp. in the waters north of Australia (0.65 to 13.1 nmol P
µg chl a
-1
h
-1
) a location that has much higher concentrations of DOP than found in
this study (Mulholland et al. 2002). APA reported for Trichodesmium spp. in the
76
southwest North Atlantic in late May was found to range from 0.03 to 0.24 µmol
MUF-P hydrolyzed µg chl a
-1
h
-1
(Mulholland et al. 2002) or 1 to 2 orders of
magnitude higher than the rates found in our study. Another study of
Trichodesmium spp. APA in the Red Sea using the substrate p-nitrophenylphosphate
found activities of 2.4 to 11.7 µmol p-nitrophenylphosphate hydrolyzed µg chl a
-1
h
-1
(Stihl et al. 2001).
However, this data represents maximal uptake rates as saturating
substrate concentrations were added and the experiments were conducted at 37ºC.
While these rates are not directly comparable, they suggest that Trichodesmium spp.
in the Western Tropical Atlantic near the Caribbean and possibly in the Red Sea are
substantially more P stressed than colonies from our study.
Only one study to date has measured both inorganic and organic P dynamics in
Trichodesmium spp., and this data set is limited. McCarthy & Carpenter (1979)
measured PO
4
3-
uptake kinetics of Trichodesmium spp. at one station in the Central
Atlantic at 30 °N and APA at 2 stations on the same transect. This early work
demonstrated the ability of Trichodesmium spp. to gain a large part of its P quota
from the organic pool. APA for 2 experiments, measured using the 3-0
methylumbelliferyl phosphate method, was two orders of magnitude higher, 170 and
300 nmol P µg chl a
-1
h
-1
,
than found in the present study. The K
s
reported for
Trichodesmium spp. (9 µM) was far greater than found in our study. A subsequent
study of P uptake in exponentially growing batch cultures of Trichodesmium spp.
77
isolated from the North Atlantic found a much lower K
s
value of about 0.4 µM for
both P replete and P deplete cells (Fu et al. 2005), very similar to the values found in
our study. The doubling time for Trichodesmium spp. colonies in the North Atlantic
based on PO
4
3-
uptake alone was 5000 hours, or 208 days (McCarthy and Carpenter
1979). A recent publication contends that this doubling time was too large by three
orders of magnitude (Moutin et al. 2005). Close inspection does show that the PO
4
3-
uptake values reported in the text (used to calculate the doubling time) differ by three
orders of magnitude from the graph of PO
4
3-
uptake kinetics. However, the authors
of the 1979 manuscript became aware of this discrepancy soon after it was
published, and state that the original values reported in the text are correct, while a
symbol was misprinted on the Y axis of the graph of this data (JM McCarthy
personal communication). Therefore, their results on the importance of DOP for
Trichodesmium spp. should still be considered relevant.
Based on the K
s
and affinity ( α) values found in our study, Trichodesmium spp.
cannot effectively compete for phosphate with smaller planktonic organisms at the
low DIP concentrations typically found in the upper water column. It does however,
have high rates of APA compared to DIP uptake. Thus we surmise that the organic P
pool is very important to Trichodesmium spp. with respect to P acquisition, while for
the nano and picoplankton in the water, it may be merely a supplement. Very similar
results were found in the central Baltic Sea where the heterocycstous cyanobacteria
78
Nodularia spumigena and Aphanizomenon sp. occur (Nausch et al. 2004). When P
limitation occurred in mid-summer in this area, 91% of PO
4
3-
uptake was carried out
by the smallest size fractions (0.2 to 3 µm) while the >10 µm fraction, which
included large amounts of cyanobacteria, was responsible for 42% of APA,
indicating DOP is a much greater source of P to heterocystous cyanobacteria in the
Baltic Sea than inorganic P (Nausch et al. 2004). The importance of the DOP pool to
Trichodesmium spp. was alluded to in a study of Trichodesmium spp. ultrastructure
in sinking and rising colonies in the Caribbean (Romans and Carpenter 1994).
Sinking colonies were found at 25 m (a depth of low PO
4
3-
concentrations) with large
inclusions of polyphosphate. Presumably Trichodesmium spp. would have had to
access the DOP pool to accumulate these large amounts of intracellular P (Romans et
al. 1994).
A very different result, however, was obtained in a recent study on PO
4
3-
uptake by
Trichodesmium spp. in the South Pacific, near New Caledonia (Moutin et al. 2005).
From P specific PO
4
3-
uptake data, the authors calculate that Trichodesmium spp.
could achieve a growth rate of 0.1 d
-1
at a PO
4
3-
concentration of 9 nM. Thus,
Trichodesmium spp. could coexist with smaller plankton by growing slowly and
using only inorganic P (Moutin et al. 2005). Our data show a very different result.
Assuming a relatively low colony P quota of 3.9 nmol (Carpenter 1983), PO
4
3-
concentrations would have to be 0.15 μM to achieve a 0.1 d
-1
growth rate. Thus, in
79
the subtropical and tropical North Atlantic, where this study was carried out,
Trichodesmium spp. does not appear to be able to grow on DIP alone. Uptake of P
from the DOP pool appears very important to growth.
It is important to consider that APA derived from the MUF-P method does not take
into account the bioavailability of the DOP pool. Björkman and Karl (2003)
estimate that 7-15% of DOP in the North Pacific is available to organisms. If the
bioavailability of DOP is 15% and this pool is constantly replenished, the importance
of DOP as a P source to Trichodesmium spp. is reduced (Table 3-3), but would still
account for over 60% of P acquisition. DOP availability of 15% reduces the
contribution of DOP in bulk plankton to a very low 2%.
To assess the volumetric contribution of Trichodesmium spp. to total uptake from the
inorganic and organic pools, we assumed a colony density in this area of 1 per liter
(Carpenter et al. 2004) to evaluate what percentage of P from each pool might make
it into Trichodesmium spp. This was calculated by dividing ambient PO
4
3-
uptake or
APA of Trichodesmium spp. by the sum of Trichodesmium spp. and bulk seawater
PO
4
3-
uptake or APA. While virtually none of the inorganic P would be taken up by
Trichodesmium spp. (0.1%), Trichodesmium spp. could be responsible for nearly
11% of the total uptake of organic P (Table 3). Therefore, Trichodesmium spp.
contributes considerably to the turnover of the DOP pool, while the turnover of the
80
DIP pool is carried out almost entirely by the smaller organisms in the water. This is
in agreement with measurement of size fractionated P uptake in the Atlantic. In the
northeastern Atlantic it was found that the smallest size fraction, 0.2 to 2 µm, was
responsible for the bulk of the PO
4
3-
uptake: 58 – 88% (Donald et al. 2001). For a
wide area of the Central Atlantic, P uptake was also found to be greatest for the
smallest organisms (Cañellas et al. 2000).
The half saturation constants calculated for the picoplankton are about equal to the
PO
4
3-
concentrations found at those same stations: 0.02 µM PO
4
3-
versus a K
s
of
0.015 µM at station B, 0.01 µM PO
4
3-
versus a K
s
of 0.015 µM at station C. These
organisms are therefore very well suited to take up inorganic P in this area, and are
operating near their maximum uptake capacity. Trichodesmium spp., on the other
hand, has a half saturation constant of 0.78 µM, much greater than the average PO
4
3-
concentration found on this cruise of 0.04 µM. Based on this data and the average
V
max
, Trichodesmium spp. is operating at 4 to 5% of maximum PO
4
3-
uptake capacity
at ambient PO
4
3-
concentrations. Thus, Trichodesmium spp. is poised to take up
pulses of high phosphate, should they come along. This finding is similar to that of
Suttle et al. (1990) which showed that increasing proportions of DIP entering the
larger size fractions as more P is added to Sargasso Sea water. At a PO
4
3-
concentration of 0.04 µM, nearly all of the PO
4
3-
(~99%), is taken up by the
picoplankton. However, if a PO
4
3-
pulse of 0.4 µM were to occur in these waters,
81
about 5% of the PO
4
3-
would enter the Trichodesmium spp. pool. At most, 15% of
PO
4
3-
could make it into Trichodesmium spp. if PO
4
3-
concentrations became high
enough. As can be seen, at nominal densities of 1 colony l
-1
, Trichodesmium spp.
will not be a large contributor to PO
4
3-
uptake even if concentrations increase;
however, PO
4
3-
would become an increasingly important portion of total P
acquisition to Trichodesmium spp. Conversely, at more extreme densities of
Trichodesmium spp. as sometimes encountered (Carpenter & Capone 1992),
inorganic P uptake could be dominated by this phytoplankter.
Using the sum of the average PO
4
3-
uptake and APA, we can calculate the turnover
time of Trichodesmium spp. colony P. Assuming a colony P content of 3.9 nmol
(Carpenter 1983), the P turnover time is about 2 days, and doubling time is 1.4 days.
This is well within estimates of colony doubling times based on C or N which range
from 1 to 2 days to over a week (Carpenter 1983, Carpenter & Romans 1991). This
does, however, assume that all of the DOP pool is bioavailable to Trichodesmium
spp. Using the estimate of Björkman and Karl (2003) of 7-15% DOP bioavailabilty,
turnover of Trichodesmium spp. colony P from DIP and DOP would be 9 – 13.5
days, and a doubling time of 6.2 – 9.4 days. Even when taking the bioavailability of
the DOP pool into account, Trichodesmium spp. appears to be able to double its
colony P in about the same amount of time as C or N based estimates of doubling
time (Carpenter 1983, Carpenter & Romans 1991). Thus, Trichodesmium spp. do
82
appear to be able to acquire most or all of the necessary P for growth from the
inorganic and organic P pools. Slower growth by Trichodesmium spp. or,
alternatively, reduced P quotas, would further decrease the doubling times of colony
P.
Picoplankton and Trichodesmium spp. both appear to be P-limited or perhaps P-
stressed in the subtropical and tropical north Atlantic, and thus the acquisition of P
by these organisms is important to their growth. It appears that each has its own
strategy to deal with this problem; picoplankton found in large numbers in bulk
water samples have a high affinity for inorganic phosphate and also the ability to
derive some of its P from the organic fraction, while Trichodesmium spp. cannot
compete very successfully for inorganic PO
4
3-
with the smaller organisms in the
water, but can obtain considerable amounts of P from the organic pool. By utilizing
this much larger pool, Trichodesmium spp. is able to coexist with picoplanktonic
organisms in this area of P limitation.
83
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87
CHAPTER IV: Phosphorus pools and cycling in the tropical North Atlantic:
Intrabasin differences and the influence of nitrogen fixation
CHAPTER IV ABSTRACT
The oceanic phosphorus (P) cycle, dominated by geological processes, is often
overlooked in biogeochemical studies. However, recent reports indicate that some
oceanic regimes are P limited; thus an evaluation of the different P pools and the
biological cycling of P is warranted in these locations. The subtropical and tropical
North Atlantic is one area proposed to be P limited. A cruise across a wide area of
the tropical North Atlantic allowed us to investigate zonal differences in
concentrations of P pools, turnover rates of the phosphate (PO
4
3-
) pool, and the
effects of the Amazon river plume in the area far off the continental shelf. The
majority of the total P pool was present as dissolved organic phosphorus (DOP,
~80%), with soluble reactive phosphorus (SRP) and particulate organic phosphorus
(POP) comprising much smaller fractions. Concentrations of both SRP and POP
increased in the Amazon River influenced areas, while DOP did not. The turnover
time of the PO
4
3-
pool was rapid on the western side of the basin (< 10 hours), but
slowed to the east (> 100 hours). Fast turnover times are indicative of P stress, and
the observed trend suggests an east to west increase in P stress in the tropical North
Atlantic. The maximal PO
4
3-
uptake rate of Trichodesmium spp., a well-studied
88
dinitrogen (N
2
) fixing cyanobacteria, also indicates this cross-basin trend in
differential P stress. We suggest that a higher rate of N
2
fixation in the western end
of the basin creates waters enriched in nitrogen (N) compared to P, and that this
influences the cross-basin differences in P stress.
INTRODUCTION
The marine phosphorus (P) cycle is a simple one, largely free of redox reactions,
dominated by the slow (geological) processes of continental uplift and weathering as
the source of P to the ocean via riverine transport, and the burial of marine organic
matter as the P sink. Internal cycling of P in the ocean is dominated by the relatively
fast biological processes of uptake from and release and degradation to the dissolved
inorganic and organic pools (Benitez-Nelson 2000). The long held view that
nitrogen (N) is the proximate and P the ultimate limiting nutrient to primary
production exists because the P source and sink terms are geological processes,
which correspond to a residence time for P in the ocean of ~ 25,000 years, while the
N pool is more dynamic, with a shorter residence time of ~ 3,000 years, because it
can respond to chemical changes in the ocean through the biological processes of
dinitrogen (N
2
) fixation and denitrification and because there is still a small amount
of PO
4
3-
remaining in the surface ocean as NO
3
-
approaches zero in the surface ocean
(Froelich 1988, Tyrrell 1999). Because P was thought to limit primary production
89
only in the long term, biological P cycling has often been overlooked. However, P is
an important macronutrient for growth (ribosomal RNA), energy generation (ATP),
information storage (DNA) and structural support (as a component of phospholipid
membranes). Additionally, new estimates of the residence time of P in the ocean
indicate that it could be as short as ~10,000 years (Benitez-Nelson 2000), and several
regions of the ocean have been shown to be P limited in the short term for at least
parts of the microbial community (Cotner et al. 1997, Thingstad et al. 2005). Thus,
it is important to quantify the different P pools in the ocean and look at the role of
biological activity in the P cycle.
The North Atlantic is one system reported to be P limited (Ammerman 2003).
Phosphate (PO
4
3-
) concentrations can be very low here (sometimes <1 nM) compared
to other areas of the ocean (e.g., ~100 nM in the North Pacific) (Wu et al. 2000) and
bacterial growth (Cotner et al. 1997) and respiration (Obernosterer et al. 2003) both
respond positively to PO
4
3-
additions. The diazotrophic cyanobacterium
Trichodesmium spp. appears to be P limited in this basin as well (Sañudo-Wilhelmy
et al. 2001, Sohm et al. submitted). The western side of the North Atlantic basin is
influenced by the Amazon River outflow, exhibiting increases in nutrients and
productivity where there is a freshwater signal (DeMaster and Aller 2001), but it is
not well known what nutrients may be limiting in river-influenced areas. N
limitation has been suggested for the river mouth and shelf areas (Demaster and Pope
90
1996), and the abundance of N
2
fixing organisms in plume influenced waters
reinforces this theory (Subramanium et al. submitted). Conversely, the prominence
of N
2
fixation in river-influenced waters could drive the system to P limitation.
While PO
4
3-
is the P form preferred by biological organisms, dissolved organic P
(DOP) concentrations are often an order of magnitude greater than PO
4
3-
(Björkman
and Karl 2000). Thus, DOP may be an important P source to some organisms,
especially under conditions of P limitation. In fact, many organisms can produce the
enzyme alkaline phosphatase, which cleaves the PO
4
3-
moiety from many organic P
molecules (Ammerman 1993). Pico- and nano-plankton in the North Atlantic have
been shown to produce this enzyme and subsequently, may get a portion of their
cellular P from the organic pool (Vidal et al. 2003). Trichodesmium spp., a globally
significant colony-forming diazotroph, appears to get a large amount of its P from
the DOP pool (Sohm and Capone 2006).
Despite the fact that P appears to be an important limiting nutrient in the North
Atlantic, few studies have quantified the different P pools in the water column or
looked at P cycling across the basin. This study was carried out on a research cruise
in the summer of 2006, where the different P pools, soluble reactive P (SRP), DOP,
and particulate organic P (POP), were quantified in surface waters, the rate of PO
4
3-
cycling was measured, and P stress in the cyanobacterium Trichodesmium spp. was
91
assessed to determine the spatial patterns in the availability of P and P stress across a
reportedly P limited areas: the tropical North Atlantic Ocean.
MATERIALS AND METHODS
Samples were collected and experiments performed on a cruise on the R/V Seward
Johnson in June and July 2006. The sampling area included the tropical North
Atlantic; leg 1 crossed from Barbados to Cape Verde between ~11 and 15º N
(stations 3-12) and leg 2 returned from Cape Verde to Barbados while traveling
down to the equator (stations 13-23, Figure 1). Samples were collected in surface
waters (3-5 m) for SRP, DOP and POP concentration analysis, uptake and turnover
rate measurements of the PO
4
3-
pool, and PO
4
3-
uptake kinetics of Trichodesmium
spp. The difference between river influenced and oceanic stations was investigated
using salinity data gathered with the CTD. Plume influenced stations are defined
here as those with salinity < 35 psu, and oceanic stations are defined as stations with
salinity >35 psu, with the exception of the equatorial station (17), which was
influenced by equatorial upwelling and thus was fundamentally different than other
oceanic stations.
Dissolved P analysis: Water was collected with a CTD rosette system, then sampled
into acid cleaned 250 mL HDPE plastic bottles, rinsed three times with sample water
92
before filling, and then frozen at -20°C until analysis on land. Current methods
cannot directly measure the ambient PO
4
3-
pool in a water sample; the molybdenum
blue colorimetric method (Strickland and Parsons 1972), routinely used by most
researchers, uses acidic conditions for the development of color and thus some PO
4
3-
is cleaved from DOP during analysis. The pool measured with this method has thus
been dubbed the soluble reactive phosphorus (SRP) pool, and has a detection limit of
~ 0.03 µM. To improve our sensitivity in oligotrophic waters, SRP was determined
using the magnesium induced coprecipitation (MAGIC) method (Karl and Tien
1992), with slight modifications. A 1M NaOH solution was added at a 1% vol/vol
ratio to 75 mL of sample water. After precipitation and centrifugation, the
supernatant was removed and the pellet resuspended in 5 mL Omni-trace clean HCl
(0.2 M). The SRP concentration was then measured with the molybdenum blue
method on a Shimadzu UV-VIS 1600 equipped with a 10 cm path length cell set to
read at 880 nm. With the concentration factor of 15 achieved by the MAGIC
precipitation step, the limit of detection is reduced to ~ 2 nM.
Total dissolved phosphorus (TDP) was determined using the high temperature
combustion and hot acid digestion method of Solarzano and Sharp (1980) to convert
organic P to PO
4
3-
, followed by the measurement of PO
4
3-
with the molybdenum blue
method. DOP was calculated by subtracting SRP and POP concentrations from the
TDP concentration.
93
Particulate Phosphorus: Samples for POP concentration analysis were collected
using a high volume water pump connected to clean tubing that was lowered over the
side of the boat to 5 m. Water was collected into polycarbonate bottles and 2-3 L
was filtered onto 25 mm pre-combusted GF/F filters using gentle vacuum (< 10 psi)
filtration. Samples were stored frozen at -20º C and then analyzed using the Hawaii
Ocean Time-Series protocol: high temperature combustion and hot acid digestion to
convert POP to PO
4
3-
followed by the measurement of PO
4
3-
with the molybdenum
blue method as described above.
Phosphate uptake: PO
4
3-
uptake was determined on water collected from the high
volume pump system. 50 mL of surface water was poured into 60 mL polycarbonate
bottles in duplicate (leg 1) or triplicate (leg 2) and 0.1-0.4 µCi of H
3
33
PO
4
added.
Samples were incubated on deck in flowing seawater at 40% ambient light for 2 – 3
hours then filtered onto 0.2 µm polycarbonate filters. A pair of controls killed with
paraformaldehyde or glutaraldehyde was run to control for abiological adsorption of
isotope. Filters were placed in 6 mL plastic ampules, and the activity was
determined using an on-board scintillation counter after addition of 5 mL of
scintillation fluid. The turnover time of the PO
4
3-
pool in a sample was calculated as
T = (R
t
*t)/(R
f
-R
k
), where t is the incubation time and R
t
, R
f
and R
k
are the
radioactivity (in counts per minute) of the total pool added, the filter and the killed
control, respectively.
94
Trichodesmium spp. PO
4
3-
uptake kinetics: Trichodesmium spp. colonies were
collected by a surface net tow with a 202 µm mesh net. Colonies were picked from
the tow into a GF/F filtered seawater (FSW) rinse with a plastic bacteriological loop
and then picked into 50 mL FSW in 60 mL polycarbonate bottles. Each bottle
contained 5-10 colonies, depending on the density of Trichodesmium in the tow, and
samples were run in triplicate with two killed controls. Unlabeled PO
4
3-
was added
to incubations in concentrations from 0-3 µM along with 0.1-0.4 µCi H
3
33
PO
4
to
generate Michaelis-Menton uptake curves. Samples were incubated for ~ 60 minutes
in on-deck incubators with flowing seawater at 40% ambient light and then filtered
onto 8 μm polycarbonate filters. The activity and uptake rate of the samples was
determined as described above for bulk measurements.
V
max
and K
s
were found by
fitting the Michaelis-Menton equation to each data set using curve fitting routines in
Sigmaplot. The kinetics of nutrient uptake are described by the equation V =
(V
max
×S)/(K
s
+S), where V is the uptake rate at nutrient concentration S, V
max
is the
maximal uptake rate (when uptake is saturated), and K
s
is the nutrient concentration
when uptake is one-half V
max
.
RESULTS
Salinity and P pool composition: Salinity in the surface waters of the tropical North
Atlantic was generally high (>35 psu; Figure 4-1). In the western North Atlantic,
95
however, salinity was lower due to inputs from the Amazon River. The influence of
the river plume on salinity could be seen at least 700 km offshore, and > 1500 km
away from the mouth of the river (Figure 4-1).
Figure 4-1 Salinity of surface waters in the tropical North Atlantic, shown in units of
ppt. Station locations and numbers are shown for reference.
Surface SRP concentrations were low at both oceanic and plume-influenced stations,
but were somewhat higher in the plume. Oceanic stations averaged 18.4 ± 5.9 nM
(range: 9.8 – 26.3) and plume-influenced stations averaged 32.6 ± 16.5 nM (range:
13.6 – 58.4 nM) (Figure 4-2a), which is a significant difference (p = 0.023).
96
Figure 4-2 Surface water P pool concentrations, in nM, in the tropical
North Atlantic: a) SRP, b) POP and c) DOP.
97
The highest SRP concentration in surface waters measured during the cruise was
64.1 nM, at the equatorial station 17.
The distribution of surface water POP was similar to that of SRP. In oceanic waters,
the POP concentration was low and ranged from 6.1 – 19 nM (average 10.2 ± 3.2
nM) (Figure 4-2b). At the equatorial station, however, the POP concentration was
notably higher, 23.7 nM. POP concentrations in plume-influenced waters were
significantly different from POP concentrations at oceanic stations (p=0.010),
ranging from 10.8 – 35.9 nM and averaging 18.2 ± 8.8 nM (Figure 4-2b). The
concentration of POP was significantly correlated to SRP concentration (R
2
= 0.60, p
= 0.042) (Figure 4-3).
PO
4
concentration (nM)
020 40 60
Particulate P concentration (nM)
0
10
20
30
40
R
2
= 0.60
Figure 4-3 The relationship of POP to SRP in surface waters of the tropical
North Atlantic for all stations where both measurements were made.
98
The concentration of DOP in surface waters was variable, but much greater than
either SRP or POP, ranging from 88 to 246 nM (Figure 4-2c). DOP concentrations
were slightly lower on average in the river-influenced stations than at oceanic
stations, 167 ± 64 nM versus 190 ± 46 nM, respectively, although this was not a
significant difference. When considering the total P pool in the tropical North
Atlantic surface waters, DOP is clearly the major form of P (figure 4-4). On
average, 84 ± 9% of P is in the DOP phase. SRP is the next most abundant form of P
(10 ± 6%), with POP making up the smallest portion (6 ± 3%).
Station
3 5 7 9 11 13 15 17 19 21 23
% of total P pool
0
20
40
60
80
100
DOP
SRP
PP
Figure 4-4 Distribution of P in the SRP, DOP, and POP pools in surface waters
of thee tropical North Atlantic.
99
PO
4
3-
uptake and turnover rates: The turnover time of the PO
4
3-
pool varied widely
across the North Atlantic, ranging from 2 to 159 h. An increase in turnover times
from west to east was found for the entire data set, but the pattern is less variable on
leg one than leg two (Figure 4-5). The turnover time was significantly correlated to
longitude (p = 0.004), which explained nearly 40% of the variation in turnover time
(R
2
= 0.39).
Longitude
-60-55 -50-45-40-35 -30-25
PO
4
turnover time (h)
0
20
40
60
80
100
120
140
160
180
R
2
= 0.39
Figure 4-5 The longitudinal gradient of PO4 turnover times in surface
waters of the tropical North Atlantic. Closed circles show data from Leg
1, open circles from Leg 2. The regression line shown is for all data
100
The maximum PO
4
3-
uptake rate in colonies of Trichodesmium spp. was also found
to vary widely across the basin, from 5.8 – 300 pmol P col
-1
h
-1
. Similar to PO
4
3-
turnover times, Trichodesmium spp. V
max
also correlated to longitude (R
2
= 0.41,
Figure 4-6).
Longitude
-60-55-50-45-40-35-30 -25
Trichodesmium V
max
(pmol P col
-1
h
-1
)
0
50
100
150
200
250
300
350
R
2
=0.41
Figure 4-6 The longitudinal gradient in the maximal PO
4
3-
uptake
rate of Trichodesmium spp.
DISCUSSION
Surface salinity and P pool composition: Mid-summer is a high flow period for the
Amazon River (REF). Thus, as expected, the effect of the river on surface salinity in
the tropical North Atlantic could be seen far away from the mouth of the river during
101
this cruise. The Amazon River is known to deliver high concentrations of nutrients
to the shelf area off Brazil, and > 90% of the river-derived inorganic N and P are
transported off the shelf to the open ocean (Demaster and Pope 1996). Indeed, the
average SRP concentration was higher in the plume than out of the plume (Figure
2a). Despite this difference, SRP values measured in this study were low at all
stations and within the ranges seen in other oligotrophic oceanic areas, and the
oceanic values were at the lower end of reported values (Table 4-1). However,
values reported in two different studies of the Sargasso Sea are much lower. Wu et
al. (2000) found SRP there to be 0.2 to 1 nM, and Cavender-Bares (2001) reported
values of 0.5 to 5 nM. These studies were both far north of our study, suggesting
that SRP in the subtropical Sargasso Sea may be consistently and considerably lower
than in the tropical North Atlantic.
Table 4-1 Phosphorus pool concentrations in surface waters of different regions of the ocean, as
reported in previous studies.
Location SRP (nM) DOP (nM) PP (nM) Study
Western Mediterranean <30-200 150-930 3-55 Tanaka et al. (2004)
North Pacific (ALOHA) <10->100 <150->300 15-17 Karl et al. (2001)
North Pacific (off Japan) 50 300 18 Suzumura et al. (2004)
North Pacific (31-48ºN) 10-1420 100-220 9-110 Yoshimura et al. (2007)
Southwest Pacific <30-60 211-281 10-25 Van Den Broeck et al. (2004)
Sargasso Sea (BATs) ~15 ~80 ~15 Ammerman (2003)
Sargasso Sea 0.2-1 74.5 - Wu et al. (2000)
Sargasso Sea 0.5-18 100 - Cavender-Bares et al. (2001)
Central Atlantic <100->250 <100-200 18-39 Cañellas et al. (2000)
Tropical North Pacific 10-64 88-246 6-36 This study
a
Range during period from June to December 2002
b
Range of data for 9-year sampling period from 1988 – 1997
c
Range during period from October 2001 to August 2002
102
Published measurements of POP concentration in the North Atlantic are few,
although the values for one study in the central Atlantic (~20 – 40 nM; Cañellas et al.
2000) and one at the Bermuda Atlantic Time series (~15 nM; Ammerman et al.
2003) were similar to those reported here. Additionally, POP concentrations were
similar to, but appear slightly lower than, those found in the North and South Pacific
and the Mediterranean (see Table 4-1). POP was higher in the plume influenced
stations, and the significant correlation found between SRP and POP measurements
(R
2
= 0.60, p < 0.001) indicates that the SRP concentration in surface waters play an
important role in determining POP concentration. This result was expected,
considering that POP is mostly P incorporated into living biomass of pico- and
nanoplankton, and PO
4
3-
is the preferred form of P for osmotrophs. However, the
SRP concentration alone can not explain POP concentrations. Standing stocks do
not indicate fluxes, and differences in demand (flux) could affect the instantaneous
SRP and POP pool concentrations. Alternatively, DOP could be acting as a source
of P for some organisms.
DOP concentrations in surface waters were slightly lower in the plume at the oceanic
stations, on average. This pattern is opposite to that seen for SRP and POP, and
could have been caused by either increased consumption or decreased production in
plume influenced waters, or low supply from the river. DOP has been shown to be
an important P source for Trichodesmium spp. (Sohm & Capone 2006), and
103
Trichodesmium spp. was found to be more abundant in the plume on leg one (Ian
Hewson, pers. comm.), leading to the intriguing possibility that Trichodesmium spp.
was drawing down DOP in surface waters of the plume. However, this difference
was not significant, so further investigation is needed to determine what controls
DOP distribution in the river-influenced area of the tropical North Atlantic. The
DOP values are, however, consistent with published values from the North Atlantic,
and also from locations around the world (Table 4-1).
DOP made up the large majority of the total P pool in surface waters, >80% on
average. Plankton produce dissolved organic matter, including DOP, in surface
waters where it accumulates because PO
4
3-
is the preferred P form (Benitez-Nelson
2000). However, because the DOP pool is so large, it may be an important source of
P for organisms that cannot compete with pico- and nano-plankton for PO
4
3-
(e.g.
Trichodesmium spp.). While few studies have measured all three components of the
P pool in oceanic waters, the relative distribution of the P pool found in this study
(~85%DOP, 10%SRP, 5%POP) appears to be consistent throughout the oligotrophic
oceans, including oceanic waters offshore of Japan (Suzumura 2004), in the North
Pacific between 30º and 40º N (Yoshimura 2007) and at station ALOHA (Karl et al.
2001), in the SW Pacific near New Caledonia (Van Den Broeck 2004) and in
Villefranche Bay in the Mediterranean Sea (Tanaka et al. 2004).
104
PO
4
3-
turnover and P stress trends across the North Atlantic basin: Shorter PO
4
3-
turnover times generally indicate a greater degree of P stress (Zohary and Robarts
1998, Flaten et al. 2005), thus it appears that plankton population on the western side
of the basin were more stressed for P during our cruise than those on the eastern side
of the basin. A survey of PO
4
3-
turnover times from different regions shows that the
turnover times in the western side of the basin are consistent with those found in
other reportedly P limited areas: the Mediterranean Sea and the Sargasso Sea (Table
4-2).
Table 4-2 PO
4
3-
turnover times in surface waters of different regions of the ocean, as
reported in previous studies.
Location Turnover time (h) Month Study
Eastern Mediterranean 2-5 May Flaten et al. (2005)
Eastern Mediterranean
(after PO addition)
94 May Flaten et al. (2005)
Eastern Mediterranean
(Levantine Basin
2-7 Jan Zohary and Robarts (1998)
Eastern Mediterranean
(Villefranche Bay)
1-91 Sep-Dec Tanaka et al. (2003)
North Pacific (ALOHA) 48-969 Year round Björkman et al. (2000)
Southwest Pacific 4-400 Year round Van Den Broeck et al. (2004)
Sargasso Sea (BATS) 9 Mar/Aug Cotner et al. (1997)
Central Atlantic 38-962 Oct/Nov Cañellas et al. (2000)
Tropical North Atlantic 2-159 Jun/Jul This study
This cross-basin trend was seen despite the fact that SRP was found to be slightly
elevated in the Amazon River plume waters in the far western basin. Thus, it is
important to state here that nutrient limitation or stress is not determined by the
105
absolute concentration of a nutrient itself, but by the relative amount of a nutrient
compared to other possible limiting nutrients (ex: Rhee 1978).
A possible explanation for this cross basin pattern is the enrichment of N relative to
P in the waters of the western basin. A limiting nutrient is generally defined as the
nutrient in least supply relative to the demand of the individuals in a community.
When A. C. Redfield observed in 1958 that the N:P ratio of particulate organic
matter was that same as the inorganic nutrient N:P ratio, his conclusion was that
marine organisms had, in fact, affected the nutrient availability, and that the
inorganic nutrient ratio reflected the demand of marine organisms. Thus, in the
marine environment, the ratio of NO
3
-
:PO
4
3-
is often cited as the line between N and
P limitation is the Redfield ratio of 16:1. While the surface water NO
3
-
:PO
4
3-
ratio
could not be determined – NO
3
-
was below the standard detection limit and no low
level method was used – the DIN:SRP ratio in the subsurface was substantially
higher than 16:1 in the western basin, decreasing somewhat linearly to less than 16:1
at the most easterly stations (Figure 4-7). This matches the pattern of turnover times
seen across the basin, with the fastest (i.e. most stressed) times in the west and
relatively slow times in the east. Slow turnover times in the eastern basin and a
DIN:SRP ratio close to or less than 16:1 indicate that N is the limiting nutrient on the
eastern side of the transect. N limitation of surface phytoplankton growth has indeed
been shown in the eastern tropical North Atlantic (Mills et al. 2004).
106
Longitude
-60 -50 -40 -30
DIN:SRP
0
10
20
30
40
50
60
70
80
Figure 4-7 The longitudinal change in DIN:SRP in the tropical
North Atlantic at 130-160 m ( z ) and at 200 m ( { ). The regression
of each data set is shown, along with the Redfield N:P ratio of 16:1
Michaels et al. (1996) and Gruber and Sarmiento (1997) hypothesized that
subsurface imbalances in the DIN:SRP ratio compared to the Redfield ratio were
caused by N
2
fixation and denitrification; N
2
fixation enriches organic matter in N
without a concurrent increase in P whereas denitrification depletes N without
depleting P. Thus, it appears likely that elevated N
2
fixation rates and the subsequent
remineralization of diazotroph inputs in surface waters leads to the accumulation of
DIN and SRP in ratios >16:1 in subsurface waters. The abundance of
Trichodesmium spp. on leg one of our cruise was highest in surface waters at stations
3, 4, 5, and 6 on the western side of the basin, while eastern stations 9, 10, and 12
107
had nearly zero colonies throughout the water column (Ian Hewson pers. comm.).
Using a video plankton recorder, Davis and McGillicuddy (2006) showed that water
column integrated Trichodesmium spp. abundance was more than five times greater
in the western than eastern North Atlantic, although this transect was at a higher
latitude (~30ºN) than our cruise. On a similar cruise track to ours, Carpenter et al.
(2004) found Trichodesmium spp. abundance along 15ºN to be highest west of
~40ºW, and also high at ~5ºN between 30 and 40ºW. N
2
fixation rates were also ~ 2-
3 times greater in these areas of high abundance than areas of low abundance
(Capone et al. 2005). In fact, N
2
fixation rates by Trichodesmium have been
measured in the western basin on numerous cruises, and while there is considerable
range to the data, rates can sometimes be upwards of 1 nmol N m
-2
d
-1
(Capone et al.
2005). Combining all the N
2
fixation rate data from Capone et al. (2005; taken from
supplemental Table 1), which includes 6 cruises, N
2
fixation by Trichodesmium spp.
averaged 247 µmol N m
-2
d
-1
to the west of 40ºW and 100 µmol N m
-2
d
-1
to the east
of 40ºW. The western basin average is a fairly robust number, as ~90% of all the
data was collected there and includes measurements from all four seasons, while the
eastern basin average is from one cruise with many fewer data points and is thus a
much more uncertain value. These data sets, taken together, show that there is some
consistent factor across the tropical and subtropical North Atlantic that promotes
Trichodesmium spp. on the western side of the basin.
108
Trichodesmium spp. is not the only N
2
fixer present in oligotrophic waters.
Unicellular diazotrophs have recently been shown to fix N
2
in surface waters –
sometimes at very high rates (Montoya et al. 2004) – and a few studies have
undertaken to measure N
2
fixation by unicellular diazotrophs in the North Atlantic.
Montoya et al. (2007) combined three data sets of N
2
fixation by small diazotrophs in
the North Atlantic, and found a pattern opposite that seen in Trichodesmium spp.: 23
µmol N m
-2
d
-1
in the western basin (>40ºW) and 72 µmol N m
-2
d
-1
in the eastern
basin (<40ºW). Again, the average value for the western basin is somewhat robust,
representing three different cruises in winter, summer and fall, while the eastern
basin values are from one cruise only and may not be representative of the system on
an annual basis. If this pattern is in fact robust, it is intriguing that the organisms
responsible for N
2
fixation change across the North Atlantic, and it remains to be
seen what factors cause this cross basin shift.
Finally, in the far western basin there can sometimes be extremely high rates of N
2
fixation over a widespread area from blooms of diatom diazotroph associations that
are stimulated by silicate inputs from the Amazon River (Carpenter et al. 1999,
Subramanian et al. submitted). This seasonally important input is, by nature,
restricted to the western basin. Thus, current data shows that N
2
fixation (in total) is
higher in the western basin than the eastern basin, but questions still remain as to the
magnitude and distribution of N
2
fixation in the eastern North Atlantic.
109
PO
4
3-
affinity: While PO
4
3-
pool turnover is considered an indicator of P limitation
(Zohary and Robarts 1998), it is important to recognize that the turnover time is
related to both biological demand and biomass. If the turnover time is high simply
because there is high biomass in the water (i.e. the demand of each individual cell is
low), fast turnover times do not indicate P limitation. The affinity ( α) of the
microbial community for PO
4
3-
indicates biomass specific demand and can be
calculated as α = 1/(T*B), where T is the PO
4
3-
turnover time and B is the biomass P
(see Thingstad and Rassoulzadegan 1999 for discussion). The affinity constant is
higher when cells are limited for P, and approaches a theoretical maximum where
uptake is limited by the diffusion of PO
4
3-
molecules to the cell surface and every
molecule that hits the cell surface is taken up. The affinity of the microbial
community for PO
4
3-
is much higher when turnover time is low (Table 4-3),
indicating that the populations at stations with the fastest turnover times are, in fact,
most P stressed.
Table 4-3 The relationship of PO
3-
4
turnover times with affinity for PO
3-
4
( α).
Values in parentheses are the standard error of the average.
Turnover time (h) α (l nmol P
-1
h
-1
) # of measurements
<10 0.021 (0.005) 4
10-20 0.0075 (0.0009) 5
20-60 0.0018 (0.0006) 5
60-160 0.0010 (0.0002) 5
110
The affinities calculated here for the more P stressed stations compare with those in
strongly P limited Mediterranean waters. The affinity of the 0.2 – 0.6 µm and 0.6 –
2 µm fraction averaged 0.011 and 0.068 l nmol P
-1
h
-1
, respectively (Tanaka et al.
2003), compared to an average of 0.021 l nmol P
-1
h
-1
for stations with turnover time
<10 hours. Thingstad and Rassoulzadegan (1999) estimated the maximum PO
4
3-
affinity of a 1µm, diffusion limited cell to be 0.046 l nmol P
-1
h
-1
; a value that
decreases with increasing cell size. Because our measurements deal with a
community of mixed sizes, it is difficult to calculate what its maximum affinity is,
however, with the average α of 0.021 l nmol P
-1
h
-1
when turnover time is <10 hours,
it appears that PO
4
3-
uptake at these stations is close to diffusion limitation, where
PO
4
3-
uptake is as efficient as physically possible.
Trichodesmium spp. P stress: N
2
fixation by Trichodesmium spp. alone is thought to
represent a significant source of new N to surface waters, and N
2
fixation rates from
Trichodesmium spp. in this region are comparable to the diffusive flux of NO
3
up
from the subsurface in this region (Capone et al. 2005). Thus it is important to
understand what factors may limit Trichodesmium spp., and studies suggest that P
may indeed limit N
2
fixation in the western North Atlantic (Sañudo-Wilhelmy et al.
2001, Krauk et al. 2006).
111
The maximal PO
4
3-
uptake rate (V
max
) by Trichodesmium spp. has been shown in
culture to increase by an order of magnitude under P depletion compared to P replete
cultures (Fu et al. 2005), and has been used in the field as an indicator of P stress
(see Chapter II). V
max
was higher on the western side of the basin, decreasing to the
eastern side of the basin, indicating P stress of Trichodesmium spp. colonies in the
western side of the basin. These results reinforce the conclusion of increased P stress
in the west inferred from PO
4
3-
turnover times. Thus, the western basin P stress of
the total community appears to extend also to the diazotrophs. This is consistent
with the assertion that N
2
fixation will continue until P limitation occurs (Redfield
1958, Tyrell 1999). Through the addition of N to surface waters, N
2
fixers will
increase the competition for P between all osmotrophic organisms, therefore
inducing P stress in themselves.
112
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116
CHAPTER V: Synthesis and conclusions: The connection of the phosphorus cycle to
diazotrophs and nitrogen fixation
INTRODUCTION
In an ocean where nitrogen (N) is considered the primary limiting nutrient,
diazotrophs should flourish and dinitrogen (N
2
) fixation should be a prominent
process. Yet this is not the case, so globally, something must be limiting N
2
fixation.
Both phosphorus (P) and iron (Fe) have been hypothesized to limit N
2
fixation in
different areas of the ocean, but a definitive answer as to which is limiting where,
based on field bioassays, has not yet been reached. Additionally, the spatial
variability of P limitation in areas where it is known to occur is not well mapped and
the relationship of P cycling to the prevalence of N
2
fixation is not known. The aim
of this stduy was to examine the relationship between P, N
2
fixers, and N
2
fixation.
SUMMARY OF RESULTS
Chapter II demonstrated that Trichodesmium spp. is more P stressed in the tropical
North Atlantic compared to the subtropical North Pacific or waters north of
117
Australia. Two common indices for P stress, maximal PO
4
3-
uptake rate (V
max
) and
alkaline phosphatase activity (APA) were used to measure the relative amount of P
stress in colonies of Trichodesmium. V
max
was at least 4 times greater in the North
Atlantic than in the North Pacific or north of Australia, when normalized to both
colony chl a content and colony trichome number, while APA was more than 2 fold
greater in the North Atlantic than Pacific. SRP concentrations in the waters north of
Australia were significantly greater than in the North Atlantic or North Pacific,
providing a simple explanation as to why Trichodesmium spp. from this area was not
seen as P stressed. Interestingly, though, SRP concentrations in the North Atlantic
and North Pacific were statistically indistinguishable. If it is not simply lower SRP
concentrations, what then is driving the North Atlantic Trichodesmium spp. to P
stress? SRP:dFe ratios strongly suggest that an abundance of Fe in the North
Atlantic where this study was carried out, supplied by Fe-rich aerosol dust or the
Amazon River plume, is promoting P limitation. Fe delivery to the remote NPSG is
far less than in the North Atlantic and concentrations are lower (Wu et al. 2000,
2001), suggesting that in the NPSG, Fe is a major control on Trichodesmium spp..
Chapter III investigated the ability of Trichodesmium spp. to compete for inorganic P
with other, smaller, planktonic organisms. This study was carried out in the
subtropical and tropical North Atlantic, an area demonstrated in Chapter II and
118
several other studies to have P limited Trichodesmium spp.. Thus, the competition
for P is critical here. Chapter III demonstrated that Trichodesmium spp. is not a good
competitor for inorganic P when compared to pico- and nano-plankton; the affinity
for PO
4
3-
of bulk water samples was more than an order of magnitude greater than in
Trichodesmium spp. Additionally, pico- and nano-plankton account for most of the
chl a normalized PO
4
3-
uptake and APA in these waters. DOP appears to supply at
most 25% of total P uptake to pico- and nano-plankton (estimated as APA/( PO
4
3-
uptake+APA)), while DOP could account for more than 90% of total P uptake of
Trichodesmium spp. from the DIP and DOP pools. Calculations suggest that
Trichodesmium spp. could not get all the necessary P for growth from DIP alone, but
when the uptake of P cleaved from DOP compounds is taken into account,
Trichodesmium spp. can grow within the range of carbon (C) and N based growth
rates seen for field populations. Thus, the DOP pool is an important one for
Trichodesmium spp., and not as much for pico- and nano-plankton, and provides a
means for them to coexist in P limited waters.
In Chapter IV, the Amazon River was investigated as a source of P to the P limited
North Atlantic, and P stress across the basin was investigated using PO
4
3-
turnover
times. SRP concentrations were low across the entire basin compared to other
locations, but were significantly elevated in plume waters, as were particulate P
119
concentrations, and these were positively correlated. DOP, on the other hand, was
slightly lower in plume waters. Given that Trichodesmium spp. uses the DOP pool
as an important P source (see Chapter III), and Trichodesmium spp. abundance was
higher in the plume, this may have been caused by exploitation of the DOP pool by
this important diazotroph. Despite the fact that the Amazon River appears to be a
source of P to the tropical North Atlantic, PO
4
3-
turnover times were very fast (a few
hours) on the western side of the basin (indicative of P stress, see below), and
increased eastward to many days. This could be due to an enrichment in surface
waters of NO
3
-
relative to PO
4
3-
above the Redfield ratio of 16:1. Indeed, DIN:SRP
in subsurface waters was ~60 in the western basin and decreased to about 16 or
lower in the eastern basin. An enrichment in N without a concurrent enrichment in P
may be a result of N
2
fixation, and published data does indicate that Trichodesmium
spp. is more abundant and active in the western basin than the eastern basin (Capone
et al. 2005, Carpenter et al. 2004, Davis and McGillicuddy 2006). It is less clear if
this same pattern occurs for small diazotrophs, but current analyses indicate the trend
is opposite that seen in Trichodesmium spp. Regardless of this fact, the data are all
consistent with higher N
2
fixation in the western basin leading to an imbalance in the
DIN:SRP ratio that causes P stress in both the total plankton community and
Trichodesmium spp. itself.
120
SYNTHESIS
That Trichodesmium spp. is P limited in the North Atlantic is not a new idea. Other
studies have also provided results that indicate this (e.g. Sañudo-Wilhelmy et al.
2001). However, data that assess the status of Trichodesmium spp. with respect to P
are lacking for the North Pacific, as no experiments have been done there. Models
and studies of nutrient fields have suggested that the North Pacific diazotrophs are
Fe, rather than P limited (Wu et al. 2000, Wu et al. 2001, Moore et al. 2002, Parekh
et al. 2005), while Karl et al. (2001) suggested that the North Pacific as a whole is in
a state of P limitation. They hypothesize that an increase in stratification leading to a
drawdown of SRP over the 1990s, coupled with an increase in Trichodesmium spp.
N
2
fixation, has driven the North Pacific Subtropical Gyre (NPSG) to P limitation.
Inspection of nutrient and experimental data from the North Pacific indicates that
this area is in fact generally N limited. Inorganic N (nitrate + nitrite or DIN) is
extremely depleted in surface waters compared to SRP such that the DIN:SRP ratio
is less than one in the upper 100m of the water column (Karl et al. 2001). This
imbalance has persisted at station ALOHA, north of Oahu, for more than a decade,
including over the period of presumptive increase in stratification and drawdown of
SRP stocks. Additionally, PO
4
3-
turnover times, another indicator of relative PO
4
3-
121
stress, average 9 days in the NPSG (Björkman et al. 2000). Slow turnover times are
generally interpreted to be indicative of low P stress. In comparison, PO
4
3-
turnover
times in the Mediterranean, where P limitation has been experimentally shown
(Thingstad et al. 2005), are on the order of hours (Zohary and Robarts 1998, Tanka
et al. 2004, Flaten et al. 2005). Culture studies have shown that Trichodesmium spp.
has a very plastic particulate N:P ratio, and that it increases dramatically after P
depletion. Particulate N:P of Trichodesmium spp. collected in the NPSG averages
less than 40, while it can be upwards of 60 in the western North Atlantic (Krauk et
al. 2006). A study of the production of nifH transcripts by unicellular diazotrophs in
the NPSG showed no increase in the number of transcripts produced when PO
4
3-
was
added to incubations (Zehr et al. 2007). As nitrogenase production was not up-
regulated, this indicates that unicellular diazotrophic N
2
fixation was not P limited.
Finally, an experiment testing the response of bacteria to N additions in the NPSG
showed that growth increased when NH
4
+
or NO
3
-
was added, an indication that
bacteria are N limited there (Donachie et al. 2001).
My data set is the first to look for P stress in Trichodesmium spp. in the North Pacific
and compare it to colonies in an area known to be P limited – the tropical western
North Atlantic. The data show that Trichodesmium spp. is not P stressed in the
NPSG or in the waters north of Australia. This begs the question: could the non-
122
diazotrophic population be P limited if diazotrophs are not? This is not likely.
Considering that the data available so far indicate that the North Pacific is, in fact,
not P limited, further studies should focus on the possibility of Fe as a more
important factor in regulating N
2
fixation there. Dissolved Fe concentrations are
extremely low in the NPSG (Brown et al. 2005), leading to an SRP:dFe ratio that is
much higher than seen in other areas; Fe is much more depleted there, relative to
SRP, setting up the conditions for Fe limitation of diazotrophs. Considering that
desertification is increasing in China (see Yang et al. 2005 for overview), an
important source of dust to the North Pacific (Sun et al. 2001), and could thus
contribute to greater dust inputs to the North Pacific in the future, it is important that
we understand if Fe is currently limiting N
2
fixation in the North Pacific, the
magnitude of that limitation, and the possible response to increased Fe inputs, so that
we know what possible feedbacks may occur in the future. It is possible that this
system is one where the feedback among dust, N
2
fixation and CO
2
uptake proposed
by Michaels et al. (2001) could be active.
While nutrient stress indicators have proved useful in this case for teasing out where
(and where not) Trichodesmium spp. is stressed, the real interest is in what the N
2
fixation response to changing conditions will be and how strong that response will
be. The most direct way to asses this is nutrient addition bottle incubations.
123
However, with Trichodesmium spp. at least, it is very difficult to keep them alive in
bottles for periods long enough to measure changes in activity. Maybe the next step
is large volume incubations of unconcentrated water to reduce bottle effects, coupled
with quantification of species specific changes in transcription of the nitrogenase
gene, nifH. If the N
2
fixation rate per copy number of nifH could be experimentally
determined, this type of incubation could yield the type of quantitative information
we are looking for.
Whether or not P is limiting to diazotrophs throughout the worlds oceans, surface
water concentrations of PO
4
3-
are very low over the geographic range Trichodesmium
spp., especiallu in the North Atlantic (Wu et al. 2000). The large size of colonies
and high total P content (compared to other phytoplankton) suggests that they should
not be able to take up enough PO
4
3-
in surface waters to grow. This realization led to
the suggestion that Trichodesmium colonies, weighed down with carbohydrate
ballast accumulated in surface waters, might sink down to the phosphocline and take
up enough PO
4
3-
there to support a number of doublings upon return to the near
surface (Karl et al. 1992). This process is termed P-mining, and was originally used
to explain some anomalous month to month changes in SRP profiles at station
ALOHA and the fact that a Trichodesmium colony represents a concentration of a
large amount of P into a relatively small package in an oceanic desert, and was not
124
based on field observations. Attempts to verify this hypothesis were unsuccessful;
colonies collected in the NPSG near the surface and near the phosphocline were
separated into sinking and rising colonies and their particulate N:P ratios analyzed
with the thought that sinking colonies should be depleted in P relative to N and
colonies rising from the phosphocline should be enriched in P. The results were
inconclusive (Letelier et al. 1998).
Trichodesmium colonies do, in fact, exhibit daily vertical migration due to
carbohydrate ballasting, but would need to travel over 100 meters to reach the
phosphocline. Villareal and Carpenter (2003) carried out a detailed experiment
investigating the diel cycle of colony vertical migration and the carbohydrate content
of sinking and rising colonies. Considering that Trichodesmium colonies will start to
consume their carbohydrates as soon as they sink deep enough to where light levels
do not support CO
2
fixation at an appreciable rate, Villareal and Carpenter (2003)
used their data to estimate how far a colony could sink before it consumed enough
carbohydrate to became buoyant again. The maximum depth a colony could sink to,
based on their estimation, was 55 m. Even if they began their migration at 15 m, the
typical location of the Trichodesmium spp. density maximum, a colony could sink to
70 m at most, whereas the phosphocline in the oligotrophic oceans is generally
greater than 100 m deep (Karl et al. 2001). This estimate was based on field
125
measurements of Trichodesmium respiration, however, the range in respiration rates
was not considered. Thus, there may be some colonies with lower respiration rates
that could sink depper in the water column than the estimate by Villareal and
Carpenter (2003). Trichodesmium erythraeum could theoretically not make it to the
phosphocline, if it were deep, as its gas vesicles are predicted to burst at 120 m
(Walsby 1992). Interestingly, a recent model of Trichodesmium spp. vertical
migration showed that ~19% of colonies could migrate down to the phosphocline
and back to the surface (White et al. 2006); however, this has still not been verified
in the field. In addition, if this phenomenon is occurring, it is important to know if it
happens with enough frequency to be an important source of P to Trichodesmium
spp. or a shuttle that brings appreciable amounts of P to the surface.
It is true that Trichodesmium spp. cannot get enough P from the SRP pool alone at
the concentrations seen in surface waters of the oligotrophic ocean, especially
considering that it must compete with pico- and nanoplankton that have a much
greater affinity for PO
4
3-
(see Chapter III). However, the DOP pool can provide 60-
90% of the total P acquired from both inorganic and organic pools. The importance
of the DOP pool to Trichodesmium spp. is apparent, but just how important depends
on the bioavailability of the DOP pool, an unknown value. Considering that DOP
may be an important source of P to osmotrophic organisms, especially,
126
Trichodesmium spp., it is imperative to understand just how important a contribution
it makes and if spatial patterns in the utilization of this pool exist. If DOP is a
substantial source of P to diazotrophs, then this pool would be contributing to new
production. What then are the implications of this to our understanding of the
stoichiometric balance of the ocean? What does this mean for the Redfield view of
the ocean? These questions need to be investigated further.
The bioavailability of the DOP pool could be measured in a number of different
ways. The decrease in DON and DOP from surface waters to subsurface should
arise mainly from biological use of these pools, and indicate how bioavailable each
pool is. If DOP is used at a greater rate than DON, this imbalance may mean DOP is
contributing to diazotroph growth. Alternatively, the natural bacteria in the ocean
can be exploited to demonstrate the bioavailability of the DOP pool. In a method
described by Naucsh and Nausch (2004), a seawater sample is filtered through a <1
µm filter such that only the bacteria remain in the sample. The seawater is then
enriched with large amounts of organic C and fixed N and allowed to grow out,
essentially forcing the bacteria to make use of all the DOP in the water that they can.
This amount of bioavailable DOP would not necessarily be the amount used in situ
by bacteria, merely the amount that could be used under extreme P depletion.
Finally, if DOP is an important P source in the oligotrophic oceans, and particularly
127
to diazotrophs, it is important to gain a greater understanding of the cycling and
turnover of this pool: production and consumption could be estimated with
radioisotopic tracer methods.
The bioavailability of the DOP pool may be dependent on the composition of the
pool. DOP is made up of different types of compounds, including mono-, di- and tri-
esters and phosphonates – direct C-P bonds – and sequence data from
Trichodesmium IMS 101 shows that they have the capability to utilize phosphonates
(Dyhrman et al. 2006). This appears to be a rare ability, as Trichodesmium spp. is
the only species of the currently sequenced cyanobacteria containing the C-P lyase
pathway. Thus, phosphonates may be another important alternative method that
Trichodesmium spp. employs to acquire P. It is difficult to measure the quantity of
the different bond classes – so far the only method is to ultrafilter DOP and analyze
the concentrated, high molecular weight DOP (termed UDOP) bonds using
31
P-
NMR. Using this method, researchers have found that ~75% of the UDOP is mono-
and di-ester bonds, and ~25% is made up of phosphonates bonds (Kolowith et al.
1999, Sannigrahi et al. 2006), and that this distribution appears to be global.
Interestingly, the source of phosphonates in seawater is unknown;
31
P-NMR analysis
failed to detect phosphonate bonds in particulate matter (Kolowith et al. 2001,
Sannigrahi et al. 2006), suggesting that phosphonates are very refractory compounds
128
that have accumulated in the ocean over a long period of time. Alternatively,
ultrafiltration could be selectively enriching phosphonates; there is no way to know
this until a method is developed to quantify DOP compounds in unconcentrated
samples.
It appears that the P cycle may be more dynamic and spatially variable than
previously recognized. Additionally, in the western North Atlantic, at least, it is
strongly linked to the N cycle through the importance of P in limiting N
2
fixers.
Ironically, it appears that N
2
fixation itself may be driving the western side of the
North Atlantic to P limitation by enriching waters with N relative to P. This would
increase demand, and thus competition, for P, leading the entire osmotrophic
community to P stress.
Current data suggest that Trichodesmium spp. is much more abundant in the western
than eastern basin, while small diazotrophs, although not as numerically important,
appear somewhat more active in the eastern than western basin. This distribution is
intriguing, and the cause unclear; chemical or physical factors might be important.
Trichodesmium spp. abundance has been linked to physical phenomenon like
anticyclonic eddies (Davis and McGillicuddy 2006) and chemical phenomenon like
large Fe inputs through dust storms (Lenes et al. 2001). Even less is known about
129
what controls unicellular diazotroph distribution. To get a better understanding,
much greater converge both spatially and temporally in Trichodesmium spp. and
unicellular N
2
fixation measurements are necessary, along with controlled laboratory
culture experiments with the key diazotrophs.
Finally, because the N, P, and Fe cycles can all be linked through the process of N
2
fixation, it is important to carry out research with this awareness, taking all of these
complex interactions into account. Future research questions include:
• What level of control does Fe exert on N
2
fixation in the North Pacific
Ocean? What is the potential for a CO
2
uptake feedback?
• How important is DOP in supporting diazotrophic growth? Are there species
specific differences between Trichodesmium spp. and the unicellular
diazotrophic cyanobacteria (Crocosphaera and the as yet uncultured group A)
in P nutrition?
• Do diazotrophs affect the distribution of DOP in the ocean?
• What, exactly, is the composition of the DOP pool and which compounds are
bioavailable to different organisms, both diazotrophic and non-diazotrophic?
130
• What are the ultimate external factors that force N
2
fixation in the world’s
oceans? What drives the apparent cross basin difference in the North
Atlantic, specifically?
131
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Abstract (if available)
Abstract
Nitrogen (N2) fixation is an important component of the marine nitrogen (N) cycle, as it provides a major source of new N to the oligotrophic ocean, which often N limited. This input could increase global N inventories, leading to the sequestration of atmospheric CO2 via the biological pump. Any changes in N2 fixation can therefore theoretically change the carbon (C) sequestration capacity of the ocean. N2 fixing organisms, like the filamentous cyanobacterium Trichodesmium spp., are inherently not N limited. One would thus expect diazotrophs to thrive in the oligotrophic oceans
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Asset Metadata
Creator
Sohm, Jill Autumn
(author)
Core Title
The connection of the phosphorus cycle to diazotrophs and nitrogen fixation
School
College of Letters, Arts and Sciences
Degree
Doctor of Philosophy
Degree Program
Biology
Publication Date
11/30/2007
Defense Date
10/16/2007
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
diazotrophs,nitrogen fixation,OAI-PMH Harvest,phosphorus cycle,Trichodesmium
Language
English
Advisor
Capone, Douglas (
committee chair
), Caron, David (
committee member
), Fuhrman, Jed Alan (
committee member
), Hammond, Douglas E. (
committee member
), Michaels, Anthony (
committee member
)
Creator Email
sohm@usc.edu
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https://doi.org/10.25549/usctheses-m955
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UC1196897
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etd-Sohm-20071130 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-595446 (legacy record id),usctheses-m955 (legacy record id)
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etd-Sohm-20071130.pdf
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595446
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Dissertation
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Sohm, Jill Autumn
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
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Libraries, University of Southern California
Repository Location
Los Angeles, California
Repository Email
cisadmin@lib.usc.edu
Tags
diazotrophs
nitrogen fixation
phosphorus cycle
Trichodesmium