Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
Effects of global change on the physiology and biogeochemistry of the N₂-fixing cyanobacteria Trichodesmium erythraeum and Crocosphaera watsonii
(USC Thesis Other)
Effects of global change on the physiology and biogeochemistry of the N₂-fixing cyanobacteria Trichodesmium erythraeum and Crocosphaera watsonii
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
EFFECTS OF GLOBAL CHANGE ON THE PHYSIOLOGY AND
BIOGEOCHEMISTRY OF THE N
2
-FIXING CYANOBACTERIA
TRICHODESMIUM ERYTHRAEUM AND CROCOSPHAERA WATSONII
By:
Nathan Samuel Garcia
____________
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOLOGY)
December 2012
Copyright 2012 Nathan Samuel Garcia
ii
Epigraph
Too much of anything is not good.
iii
Dedication
I dedicate this dissertation to the many forefathers and mothers who have carried us
through this stitch in time and to all who defend them.
iv
Acknowledgements
I would like to thank my committee members, friends and family members for their
support throughout this study. I would also like to thank the University of Delaware, the
University Southern California and the National Science Foundation for providing
financial assistance for this research.
v
Table of Contents
Epigraph ii
Dedication iii
Acknowledgements iv
List of Tables vii
List of Figures viii
Abstract xi
Preface xiv
Contributions of Authors xxxvi
Introduction 1
References in the Introduction 11
Chapter 1: Interactive effects of irradiance and CO
2
on CO
2
- and
N
2
fixation in the diazotroph Trichodesmium erythraeum
(Cyanobacteria) 16
Chapter 1 Abstract 19
Chapter 1 Introduction 22
Chapter 1 Methods 26
Chapter 1 Results 33
Chapter 1 Discussion 37
Chapter 1 Acknowledgments 46
Chapter 1 References 47
Chapter 1 Tables 54
Chapter 1 Figures 55
Chapter 2: Combined effects of CO
2
and irradiance on large and small
isolates of the unicellular N
2
-fixing cyanobacterium
Crocosphaera watsonii from the western tropical Atlantic
Ocean 61
Chapter 2 Abstract 63
Chapter 2 Introduction 65
Chapter 2 Methods 68
Chapter 2 Results 74
Chapter 2 Discussion 80
vi
Chapter 2 Acknowledgments 88
Chapter 2 References 89
Chapter 2 Tables 94
Chapter 2 Figures 96
Chapter 3: Co-limitation interactions between phosphorus, light and
CO
2
in the unicellular photosynthetic diazotroph
Crocosphaera watsonii 104
Chapter 3 Abstract 105
Chapter 3 Introduction 106
Chapter 3 Methods 108
Chapter 3 Results 112
Chapter 3 Discussion 116
Chapter 3 References 123
Chapter 3 Tables 128
Chapter 3 Figures 129
Chapter 4: Summary and Conclusions 136
Comprehensive List of References 144
vii
List of Tables
Chapter 1 Table 1: Carbonate system measurements and calculations
for light-CO
2
experiments with Trichodesmium
erythraeum 54
Chapter 2 Table 1: Outline of experiments with Crocosphaera
watsonii (WH0401 and WH0402) 94
Chapter 2 Table 2: Carbonate system measurements for light-CO
2
experiments with Crocosphaera watsonii
(WH0401 and WH0402) 95
Chapter 3 Table 1: Carbonate system measurements for
phophorus-light-CO
2
experiment with
Crocosphaera watsonii (WH0003) 128
Chapter 3 Table 2: Best fit response kinetics as a function of
Irradiance for Crocosphaera watsonii
(WH0003) 129
Chapter 3 Table 3: Best fit response kinetics as a function of
phosphorus for two pCO
2
levels and two light
levels for Crocosphaera watsonii (WH0003) 130
viii
List of Figures
Preface Figure 1: The oceanic carbon cycle xxx
Preface Figure 2: The great ocean conveyor belt (from Broecker
1987) xxxi
Preface Figure 3: Concentrations of atmospheric CO
2
over the past
420,000 years as recorded from air bubbles
trapped ice cores above Lake Vostok, Antarctica
(modified from Petit et al. 1999) xxxii
Preface Figure 4: The marine nitrogen cycle (modified from
Capone 2000) xxxiii
Preface Figure 5: Nitrate concentrations as a function of depth from
the ocean surface xxxiv
Preface Figure 6: Regions of major oxygen minimum zones
(OMZ’s) in the world’s oceans (shaded areas
represent OMZ’s; from Kamykowski and Zentara
1990) xxxv
Chapter 1 Figure 1: Combined effects of irradiance and pCO
2
on
growth of Trichodesmium erythraeum (IMS101
and GBRRLI101) 55
Chapter 1 Figure 2: Combined effects of irradiance and pCO
2
on CO
2
-
and N
2
-fixation by Trichodesmium erythraeum
(IMS101) 56
Chapter 1 Figure 3: Combined effects of irradiance and pCO
2
on CO
2
-
and N
2
-fixation by Trichodesmium erythraeum
(GBRRLI101) 57
Chapter 1 Figure 4: Combined effects of irradiance and pCO
2
on
cellular carbon, nitrogen and phosphorus quotas
of Trichodesmium erythraeum (IMS101) 58
Chapter 1 Figure 5: Combined effects of irradiance and pCO
2
on
gross:net N
2
-fixation rate ratios and trichome
length of Trichodesmium erythraeum (IMS101) 59
ix
Chapter 1 Figure 6: Postulated effects of elevated pCO
2
on N
2
fixation
by Trichodesmium erythraeum (IMS101 and
GBRRLI101) in a water column setting 60
Chapter 2 Figure 1: Effects of irradiance on growth, CO
2
- and
N
2
-fixation rates, cell diameter, and gross:net
N
2
-fixation rate ratios of Crocosphaera watsonii
(WH0401 and WH0402) 96
Chapter 2 Figure 2: Effects of pCO
2
on growth, gross:net
N
2
-fixation rate ratios, and cellular gross N
2
-
and net N
2
-fixation rates of Crocosphaera
watsonii (WH0401 and WH0402) 97
Chapter 2 Figure 3: Effects of pCO
2
on CO
2
-fixation rates,
PN accumulation rates and gross
N
2
fixation:PN accumulation rates of
Crocosphaera watsonii
(WH0401 and WH0402) 98
Chapter 2 Figure 4: Combined effects of irradiance and pCO
2
on
growth and CO
2
-fixation rates of Crocosphaera
watsonii (WH0401 and WH0402) 99
Chapter 2 Figure 5: Combined effects of irradiance and pCO
2
on N
2
fixation and particulate nitrogen accumulation
rates by Crocosphaera watsonii (WH0401 and
WH0402) 100
Chapter 2 Figure 6: Combined effects of irradiance and pCO
2
on
gross:net N
2
-fixation rate ratios and gross:
particulate nitrogen accumulation rate ratios
of Crocosphaera watsonii (WH0401 and
WH0402) 102
Chapter 2 Figure 7: Combined effects of irradiance and pCO
2
on
N
2
-fixation rates by Crocosphaera watsonii
(WH0401) during the dark and early portion of
the photoperiod 103
Chapter 3 Figure 1: Effects of irradiance on growth, CO
2
- and N
2
-
fixation rates and cell diameter of Crocosphaera
watsonii (WH0003) 131
x
Chapter 3 Figure 2: Combined effects of phosphorus, light and pCO
2
on growth of Crocosphaera watsonii (WH0003) 132
Chapter 3 Figure 3: Combined effects of phosphorus, light and pCO
2
on CO
2
- and N
2
fixation by Crocosphaera
watsonii (WH0003) 133
Chapter 3 Figure 4: Combined effects of phosphorus, light and pCO
2
on phosphorus-uptake rates, growth:phosphorus-
uptake rate ratios and mean cellular phosphorus
quotas of Crocosphaera watsonii (WH0003) 134
Chapter 3 Figure 5: Combined effects of light and pCO
2
on cellular
phosphorus demand for CO
2
and N
2
fixation by
Crocosphaera watsonii (WH0003) 135
xi
Abstract
Approximately half of natural global biological dinitrogen (N
2
) fixation takes places in
the oceans. Estimates suggest that cyanobacteria including the filamentous genus
Trichodesmium and unicellular groups like Crocosphaera collectively contribute the
majority of oceanic N
2
fixation. Rapidly changing environmental factors such as the
rising atmospheric partial pressure of carbon dioxide (pCO
2
), shallower mixed layers
(higher light intensities) and changes in nutrient fluxes to the euphotic zone (from both
deep water and atmospheric inputs) will likely affect N
2
-fixation rates in the future ocean.
Several studies using laboratory cultures of Trichodesmium erythraeum and
Crocosphaera watsonii have documented increased N
2
-fixation rates when pCO
2
was
doubled from present-day atmospheric concentrations (~380 ppm) to 100-year projected
future levels (~750 ppm). Because marine N and C biogeochemistry are tightly linked,
this potential impact on the N cycle will likely have important consequences for the C
cycle. These findings provided impetus for examining effects of elevated pCO
2
on N
2
-
fixation rates in combination with other environmental factors like iron (Fe), phosphorus
(P), and light. Thus, I examined interactive effects of light and pCO
2
on growth, N
2
- and
CO
2
-fixation rates by two strains of T. erythraeum (GBRTRLI101 and IMS101) in
laboratory semi-continuous cultures. The effect of elevated pCO
2
on gross N
2
-fixation
rates was high in cultures (GBRTRLI101 and IMS101) growing under low (38 µmol
quanta m
-2
s
-1
) and mid irradiances (100 µmol quanta m
-2
s
-1
), but this effect was reduced
at high light (220 µmol quanta m
-2
s
-1
). This study suggests that elevated pCO
2
may have
a strong positive effect on gross N
2
fixation by Trichodesmium in intermediate and
bottom layers of the euphotic zone, but perhaps not in light-saturated upper layers of the
xii
oceans. I also examined the combined effects of irradiance and pCO
2
on growth, N
2
- and
CO
2
-fixation rates in two western tropical Atlantic Ocean isolates of C. watsonii
(WH0401 and WH0402). In both strains, cellular growth, gross N
2
- and CO
2
-fixation
rates were reduced in low-pCO
2
-acclimated cultures (190 ppm) relative to present-day
(~385 ppm) or future (~750 ppm) pCO
2
treatments. Unlike previous reports for C.
watsonii (WH8501), however, N
2
-fixation rates did not increase further in cultures
acclimated to 750 ppm relative to those maintained at present-day pCO
2
. Both increasing
irradiance (p<0.001) and pCO
2
(p<0.03) had a significant negative effect on gross:net N
2
-
fixation rates in WH0402 and trends were similar in WH0401, implying that retention of
fixed N was enhanced under elevated irradiance and pCO
2
. These results also imply that
growth rates and N
2
-fixation rates of WH0401 and WH0402 respond differently to
changing pCO
2
. These data, along with previously reported results, suggest that C.
watsonii may have wide-ranging, strain-specific responses to changing irradiance and
pCO
2
, emphasizing the need to examine a range of isolates within this genus. In the third
chapter, I examine three-way interactions between pCO
2,
P availability, and irradiance in
a Pacific Ocean isolate of C. watsonii (WH0003). First, I document P requirements for
growth, N
2
- and CO
2
-fixation rates by generating Monod functional response curves
under high and low pCO
2
and light conditions. The effect of elevated pCO
2
on these
physiological rates was greatly enhanced under low P conditions in comparison with P-
replete cultures, due to a high threshold concentration of P for these rates in low-pCO
2
-
acclimated cultures. This trend was consistent under low (40 µmol quanta m
-2
s
-1
) and
high (150 µmol quanta m
-2
s
-1
) irradiance, and suggests that the P demand for growth of
C. watsonii decreases with increasing pCO
2
. The effect of elevated pCO
2
on N
2
-fixation
xiii
rates was reduced 8-fold under low light in comparison with high-light-acclimated
cultures (p<0.05), reflecting a light-pCO
2
interactive trend opposite to that documented
for Trichdodesmium. This difference in the interactive effect of light and pCO
2
on N
2
fixation by Crocosphaera when compared with Trichodesmium might be caused by the
different N
2
-fixation strategies that they use (temporal vs. spatial separation of N
2
and
CO
2
fixation). These studies emphasize the need to examine interactive effects of
multiple environmental variables on a variety of oceanic N
2
fixers to accurately predict
effects of global change on the N and C cycles.
xiv
Preface
To begin, I would like to mention first that I am thankful for the efforts of so
many excellent writers in this field who have given great presentations of global
processes to help all students, novices and experts, understand the way the world works
from a biogeochemical perspective. With that said, this preface may seem like a non-
traditional approach to begin a dissertation, but I decided to include this section to help
readers see the overall relevance of this research and is intended to provide a brief
overview of the carbon and nitrogen cycles in the oceans. To begin, close your eyes for
15 seconds and imagine the earth as a living, breathing organism. Go ahead, close them
and envision the earth. Remember this image. After all, the surface layer of the earth is
called the biosphere. If you don’t want to close your eyes now, that’s ok because you
might feel like closing them in about 5 minutes.
A vast majority of the earth’s surface is covered by water. Water is essential for
life; without it, you and I would die. Therefore, one might think of marine biologists as
doctors of the planet. From this point of view, I hope everyone can see the importance of
the job. When I was 11 years old, I tried to drink as much water as I could in one sitting.
With good fortune, I became sick and stopped the experiment before I drank too much. I
didn’t know that drinking too much water could have killed me. But there I was, 11
years old, doing experiments with water. I had no idea I would do so many later in life.
Too much of anything is not good.
Because much of this research is centered on the concentration of atmospheric
carbon dioxide (CO
2
), I thought I would begin with a short description of the global
carbon cycle. Carbon is constantly cycled through the atmosphere, biosphere and the
xv
lithosphere. As it turns out, current estimates indicate that greater than 99.9% of all the
carbon on the earth is in locked marine sediments (Raven et al. 2005). If we exclude
marine sediments, 73% of the remaining carbon on earth exists in the world’s oceans
mostly as inorganic carbon, 26% is on the continents in either alive or dead material
including fossil fuels, and about 1% is in gaseous form within the atmosphere. You
might ask then, if atmospheric CO
2
is such a small portion of the total carbon on earth,
why, on earth, is it so important? One of the reasons that it is so important is because the
amount in the atmosphere is exactly that. It is a small amount, which means that
relatively small fluctuations can yield large percentage changes. For instance if the
percentage increased from 1% to 1.5%, although 0.5% is small compared to the total, the
percent change would be 50%. Since humans starting burning fossil fuels, the
concentration of atmospheric CO
2
has risen by about 38%. But, as you’ll see, if the
oceans weren’t involved in the carbon cycle at all, this percentage increase would be at
least twice as high.
Beginning with atmospheric CO
2
, the oceans act like a sponge in that they have
an enormous capacity to absorb CO
2
when it reacts with water (H
2
O) to form carbonic
acid (H
2
CO
3
) (Figure 1; Caldeira and Wickett 2003). You can observe the reverse of this
reaction every time you drink soda and CO
2
forms bubbles. Once carbonic acid is
formed, it quickly dissociates into bicarbonate (HCO
3
-
) and a hydrogen ion (H
+
).
Bicarbonate is the largest reserve of dissolved inorganic carbon in the ocean and its
concentration in the surface layers of the ocean (2000 µM) is 200 times larger than the
concentration of dissolved CO
2
(~10 µM). You can think of CO
2
as cash in your wallet;
every time the big boss pays you with cash, you deposit most of it into the bicarbonate
xvi
bank while only a small portion stays in your wallet. This analogy applies to the ocean.
As CO
2
dissolves from the atmosphere into the ocean, most of it reacts with water and
ultimately gets converted to bicarbonate, and only a small portion stays in the dissolved
form of CO
2
. This is how the oceans behave like an atmospheric CO
2
sponge.
Bicarbonate can further dissociate into another H
+
ion and a carbonate (CO
3
2-
) ion
(Figure 1). The dissociation of these compounds (H
2
CO
3
and HCO
3
-
) is related to the pH
of the water, which is simply a measurement of the H
+
ion concentration. Thus, adding
CO
2
decreases the pH of water; making it more acidic after H
+
ions are released from
H
2
CO
3
. For instance, part of the reason that soda decays hydroxyapatite in your teeth and
leads to cavities is because it contains CO
2
, which reacts with water to form carbonic acid
and thereby increases the acidity of soda. Since the beginning of the industrial revolution,
the oceans have absorbed approximately one third of all the CO
2
that has evolved from
human-induced burning of fossil fuels, with one third being absorbed by terrestrial
primary production and one third remaining in the atmosphere. If the oceans did not have
this ability to absorb atmospheric CO
2
, the concentration of CO
2
in the atmosphere would
be ~75% higher than it was before the beginning of the industrial revolution, ~200 years
ago.
Primary producers like phytoplankton, macro-algae and land-based plants also
remove CO
2
from the atmosphere through photosynthesis by converting it into other
carbon compounds that can be stored in the earth for years to 100’s of millions of years.
For instance, one carbon compound, calcium carbonate (CaCO
3
) is an inorganic carbon
compound that is formed by many different biological organisms including
phytoplanktonic algae, like the coccolithophore Emiliania huxleyi. This form of carbon
xvii
sinks out of the water column relatively quickly and represents the vast majority of
carbon that is buried in the ocean floor and can have an extremely long residence time in
marine sediments before returning to the atmosphere as CO
2
. Carbon bound in organic
molecules sink more slowly are respired for their energy by all sorts of marine life and as
a result, this carbon returns to CO
2
much more quickly. In comparison with CaCO
3
, a
large percentage of organic carbon does not even reach the ocean floor because it is
respired so rapidly.
Photosynthetic organisms are active in the sun-lit surface layers of the ocean
(down to 100-200 m depth) and have the ability to increase the pH of seawater in these
surface layers of the ocean by removing CO
2
, making it more basic. As algae create
carbon compounds they sink out of the water column and some are eventually buried in
marine sediments. As continental plates diverge, carbon in sediments is forced into the
earth’s mantle, combusted and CO
2
is blown out of volcanoes back to the atmosphere,
thereby completing the carbon cycle (Figure 1). In addition to biological production of
carbon compounds, CO
2
is also removed from the atmosphere by the weathering of rocks
such as limestone (also CaCO
3
); conversely, formation of limestone releases CO
2
from
bicarbonate to the atmosphere, just as the production of concrete does. Over geologic
history, these major processes have governed the carbon cycle. Changes in the rates of
any of these processes lead to changes in the relative amounts of these various forms of
carbon. As you can see, primary producers like algae, have an important role in
maintaining biogeochemical cycles like the carbon cycle and can have a large impact on
the small percentage of earth’s carbon that is in the atmosphere as CO
2
.
xviii
Climate change models predict that anthropogenic burning of fossil fuels,
production of concrete (creation of CaCO
3
releases CO
2
), deforestation and changes in
land use will cause atmospheric CO
2
to double the present-day concentration of ~390
parts per million (ppm) within the next 100 years (Raven et al. 2005; Alley et al. 2007-
IPCC). On relatively short timescales, CO
2
diffuses into the upper layers of the oceans
from the atmosphere, which are eventually mixed into deeper layers by the ocean’s
“conveyor belt” circulation over a ~1000 year cycle (Figure 2; Broecker 1987). Thus,
over long periods of time the oceans have a tremendous capacity to absorb atmospheric
CO
2
. Because ocean mixing is slow, it cannot reach its full capacity to absorb CO
2
in a
short amount of time. This is one of the reasons that the concentration of CO
2
is
increasing at an exponential rate both in the atmosphere and in the surface layers of the
oceans.
Anthropogenic sources of CO
2
have led to an increase in the partial pressure of
CO
2
(pCO
2
) in the atmosphere of approximately 106 parts per million (ppm). Before the
dawn of the industrial revolution, atmospheric air bubbles trapped in Antarctic ice (above
Lake Vostok) over the past 420,000 years indicate that pCO
2
had consistently cycled
between 190 and 280 ppm (Figure 3; Petit et al. 1999). When atmospheric pCO
2
was low
(180-190 ppm) the earth was in a “glacial maximum” period. The last glacial maximum
period was approximately 18,000 years ago. During this time, mean global temperature
was low and relatively large regions of the continents were covered with ice. When
atmospheric pCO
2
was higher (280-300 ppm) the earth was in an “interglacial” period,
mean global temperature was warmer and much of the glacial ice had melted into lakes
and oceans. The atmosphere has cycled through these glacial/interglacial states about 4.5
xix
times during the past 420,000 years and one complete cycle lasted approximately 100,000
years (Figure 3; Petit et al. 1999).
Today, pCO
2
is approximately 386 ppm, which far exceeds the natural range
under which the earth had cycled for the past half million years. Within the next 100
years, prediction models estimate that pCO
2
will be much higher than the present-day
concentration, between 750-1000 ppm pCO
2
(Raven et al. 2005). These predicted
estimates exceed natural interglacial peak pCO
2
concentrations by 170-260%. Along
with this predicted rise in atmospheric pCO
2
, the pH of the world’s oceans is expected to
decrease by approximately 0.35 units from today’s estimated mean value of ~8.2-8.25.
This may not seem like a big change, but because the pH scale is logarithmic, it is
equivalent to a 125% increase in the oceanic H
+
ion concentration and will drastically
change ecosystems and the biogeochemistry of the oceans. This process is called ocean
acidification.
Since the beginning of the industrial revolution, the production of atmospheric
CO
2
has exponentially accelerated to rates that may have never existed in the history of
the earth. This extremely high rate of change will cause dramatic changes in life on
earth, because many organisms simply cannot adapt and evolve as quickly. Many species
may become extinct as a consequence of the direct or indirect effects of this process.
This extinction process will have tremendous impacts on biogeochemical cycles like the
carbon cycle, food web dynamics and ecosystems not only in the oceans but also on land.
Calcifying marine organisms will be impacted first, such as corals, pteropods and some
species of algae like coccolithophores (Riebesell 2004; Orr et al. 2005; Raven et al. 2005;
Feely et al. 2008). This is because body structures of these organisms depend heavily on
xx
calcium carbonate (CaCO
3
), which also dissociates in water according to the pH of
seawater in a similar way to the dissociation of H
2
CO
3
and HCO
3
-
. As the oceans
become more acidic, the formation of CaCO
3
becomes more difficult. In fact, many
ocean scientists would agree that almost all coral reefs in shelf waters of the oceans
around the world will be gone within the next 50 to 100 years if we do not begin reducing
the combustion of fossil fuels immediately (Feely et al. 2008). You can imagine the
consequences for many species of life that depend solely on coral reefs for their survival.
They will also be gone.
Over the past 20 years, global warming, another effect of rising concentrations of
atmospheric CO
2
, has received a lot of attention in mainstream media and the political
world. Today, many ocean scientists would agree that ocean acidification is arguably one
of the most serious problems we have been faced with since the beginning of the human
era. Although global warming and ocean acidification are separate effects of
anthropogenic effects on the carbon cycle, they act synergistically on each other. To
understand some of the details of this interaction, let’s first consider how atmospheric
CO
2
has a greenhouse effect on warming the biosphere. First, solar radiation enters the
atmosphere as short wave radiation. This high-energy radiation easily passes through
many atmospheric gases like H
2
O vapor and CO
2
. As the earth absorbs this energy, the
continents constantly radiate it in the opposite direction as low-energy, long-wave heat
radiation. Long-wave radiation does not easily pass through many atmospheric gases.
Hence, heat is trapped and warms the biosphere. As global temperatures increase, polar
ice melts. This low-density fresh water then pours into the oceans near the poles and
floats on heavier salt water. In turn, melting ice water acts as a cap and slows the mixing
xxi
process of the oceans, thereby decreasing the CO
2
absorption rate of the deep layers of
the ocean. This reduced CO
2
absorption rate accelerates the rise in atmospheric CO
2
and
increases ocean acidification in the surface layers of the ocean. In addition, just as a wet
sponge cannot absorb as much water as a dry sponge, the ability of the surface layers of
the ocean to absorb CO
2
is constantly reduced as more and more CO
2
is continually
absorbed. Thus, as elevated atmospheric CO
2
warms the globe, both global warming and
ocean acidification together accelerate the rise in atmospheric CO
2
(Raven et al. 2005).
This accelerated warming may bring the earth’s atmosphere into a new plateau and
stabilize at a very high mean global temperature. We do not know, however, if this will
happen. Instead, this positive feedback may stop when negative feedback mechanisms
are triggered and the planet begins to freeze. This reversal has happened before, many
times throughout the earth’s recent history.
Intuitively, elevated atmospheric pCO
2
seems like a direct trigger for global
warming and the changes associated with glacial/interglacial shifts in climate, but we do
not know, with certainty, the primary cause of these shifts. For instance, some data
suggest that solar insolation might explain glacial/interglacial cycling of the atmosphere.
By this logic, climate forcing is linked to eccentricities of the earth’s orbit around the sun
(Imbrie et al. 1992; Petit et al. 1999). Although the primary cause of glacial/interglacial
climate shifts is not apparent, changing concentrations of atmospheric CO
2
are clearly
involved as either a natural cause or effect of climate change.
Because the carbon cycle shifts with changes in climate, the nitrogen cycle must
also shift with changes in climate because they are linked in biochemicals (Capone et al.
1997; Gruber 2008). In fact, some speculation suggests that the nitrogen cycle may be
xxii
partially responsible for these large fluctuations in atmospheric pCO
2
during
glacial/interglacial periods. Consider for instance that for sustainable growth, primary
producers like phytoplankton require nutrients such as CO
2
, reactive nitrogen (i.e. organic
nitrogen, NH
3
, NH
4
+
, NO
2
-
, NO
3
-
) and phosphorus. If reactive nitrogen concentrations
are too low to support growth, then the rate of biological draw down of CO
2
from the
atmosphere into oceanic biomass will be reduced. If nutrient concentrations like nitrogen
and phosphorus are high, phytoplankton can photosynthesize at optimal rates, and
thereby sequester large amounts of atmospheric CO
2
, reactive nitrogen and phosphorus as
they grow. Thus, ocean biology pumps CO
2
from the ocean surface and atmosphere,
along with nutrients from the sun-lit layers of the water column to the deep ocean through
photosynthesis, growth and sinking of carbon-rich mass (Figure 1). In this context, the
oceans act as a reservoir into which, CO
2
is biologically pumped during periods when
atmospheric pCO
2
and primary production rates are high. Likewise, CO
2
is released from
this grand carbon reservoir during periods when atmospheric pCO
2
and primary
production rates are low. Thus, the carbon and nitrogen cycles are linked in the oceans
by primary production of phytoplankton.
The nitrogen cycle is unique relative to other biological nutrient cycles in that it
has a large gaseous reservoir that is not biologically available as a growth substrate.
When reactive nitrogen concentrations in the water are low, the gaseous N
2
reservoir is
always high, representing 78% of atmospheric gas at sea level. Thus, some organisms
have developed a way to tap into the gaseous supply of nitrogen with an enzyme that
breaks the triple bond of N
2
gas by adding electrons to it (Figure 4). This enzyme is
called nitrogenase. Hence, N
2
gas gets reduced to ammonium (NH
4
+
), which can then be
xxiii
used to make glutamate, a simple amino acid. The reduction of N
2
gas is termed N
2
fixation. Glutamate can easily be converted to other amino acids, which are building
blocks for proteins and other N containing compounds that cells need to grow. Thus,
biological N
2
fixation represents a major pathway of N cycling out of the atmosphere and
into the ocean.
As phytoplankton species are consumed by higher trophic levels, organic nitrogen
circulates throughout food webs. Eventually, it is remineralized into nitrate (NO
3
-
) by
another process termed nitrification, as organisms die, decay and release NH
4
+
, the
substrate for nitrifying prokaryotes (Figure 4). In this process, organic carbon is de-
coupled from organic nitrogen and the biochemical link between the carbon and nitrogen
cycles is broken. Because dead organisms are constantly sinking, NO
3
-
concentrations are
high in deep ocean water. Likewise, NO
3
-
concentrations are low in surface waters
(euphotic zone) because living organisms are constantly taking up NO
3
-
and reducing it to
NH
4
+
to produce proteins and other N-containing biological compounds (Figure 4).
Hence, low concentrations of NO
3
-
in surface waters are indirectly driven by light
through photosynthetic primary production and biological uptake of NO
3
-
(Figure 5).
In some highly productive shelf regions of the world’s oceans, primary production
rates by phytoplankton are so high that sinking rates of organic matter are amplified. In
these regions, decaying, sinking biomass feeds organisms that live near or on the shelf
floor. As these organisms consume the sinking organic carbon, they also consume
dissolved oxygen, creating environments that are low in O
2
and rich in CO
2
. Because
oxygen is consumed during the respiration of organic carbon, these regions in the deep
water are termed oxygen minimum zones (OMZ’s) and major OMZ’s in the world’s
xxiv
oceans are accompanied by high primary production rates in overlying surface water
(Figure 6; Kamykowski and Zentera 1990).
Although one might tend to think of oxygen as essential for respiration, there are
many elements and compounds that can serve the same function. Organic carbon stores
energy in the form of electrons. Hence, energy is extracted from organic carbon in the
form of electrons. Once extracted, electrons are passed to many different compounds
within the cell and energy is transformed and stored in the cell in different ways. This
process is very similar to the way that a dam transforms kinetic energy of water into
another form of energy that can be stored. Ultimately, electrons need a final resting
place, just like water above the dam needs a place to flow into, below the dam. This is
why oxygen is important for respiration; O
2
serves as a terminal electron acceptor. In
nature, there are many terminal electron acceptors that are important for respiration in
environments where the concentration of oxygen is low. In OMZ’s, NO
3
-
is an important
electron acceptor and is denitrified when it accepts electrons during respiration (Figures
4, 5 and 6). During denitrification, NO
3
-
is converted back to N
2
gas, which can cycle
back to the atmosphere, thereby completing the nitrogen cycle. In another biological
process termed anammox, NH
4
+
and nitrite NO
2
-
are consumed to produce N
2
gas (Figure
4; Kuypers et al. 2003, 2005). Thus, both organic carbon and reactive nitrogen (NH
4
+
,
NO
2
-
and NO
3
-
) are converted back to gaseous forms in OMZ’s. Thus, these regions
function as major sites of overturn in the carbon and nitrogen cycles and are expected to
expand as a result of global change within the next 100 years (Stramma et al. 2008).
One reason that primary production rates are so high in regions that overlie
OMZ’s is that water is constantly up-welled supplying phytoplankton with high
xxv
concentrations of nutrients (Figure 6). In major OMZ’s, upwelled water is rich in CO
2
(which can be considered as being analogous to a nutrient), as well as nutrients like NO
3
-
and phosphate (PO
4
3-
). Typically, the collective community of phytoplankton takes up
carbon, nitrogen, and phosphorus at a ratio of 106:16:1, respectively (Redfield 1963). In
the absence of denitrification, this water would have a nitrogen:phosphorus ratio of 16:1
but because some of the NO
3
-
was removed by denitrification within the deep OMZ, NO
3
-
is lower than the expected value relative to phosphorus; the N:P ratio is less than 16. As
the collective phytoplankton community takes these nutrients up at a mean ratio of 16:1,
nitrogen approaches zero before phosphorus does. Hence, when upwelled water is
depleted of nitrogen at the surface, the water is rich in phosphorus (excess CO
2
is simply
out-gassed to the atmosphere in these regions). Water that is depleted of reactive
nitrogen limits the growth of many species of phytoplankton. But, because this water is
devoid of nitrogen and rich in phosphorus, species that can fix N
2
gas have a tremendous
advantage over others, in terms of growth. Nitrogen fixation in regions that have a low
N:P ratio not only serves as a point of entry for nitrogen from the atmosphere to the
ocean, but also functions to reset the N:P ratio in the water back to 16 (Capone and
Knapp 2007).
There are several taxonomic groups of N
2
fixing plankton in the oceans (Zehr et
al. 2001; Falcon et al. 2004; Zehr et al. 2008). Some of the major N
2
fixing organisms in
the oceans can also fix carbon by photosynthesis. Two genera of this group are the
cyanobacteria Trichodesmium and Crocosphaera. (Capone et al. 1997; Zehr et al. 2001)
Because they can fix N
2
and CO
2
they are important components that link the global
carbon and nitrogen cycles.
xxvi
Like any biological process, environmental factors impress limitations on N
2
fixation. One obvious limitation is the concentration of phosphorus in the water. Another
environmental factor that limits N
2
fixation in the water column is light intensity, which
is obviously depth dependent. Another major limiting factor is iron. Iron directly limits
N
2
fixation rates because iron is needed as a cofactor for the N
2
-fixing enzyme,
nitrogenase. Lastly, several recent laboratory studies have indicated that the present-day
concentration of atmospheric CO
2
probably limits N
2
-fixation rates in Trichodesmium
and Crocosphaera (Barcelos e Ramos et al. 2007; Hutchins et al. 2007; Levitan et al.
2007; Fu et al. 2008). Many of these same variables interact in similar ways to affect
CO
2
fixation by primary producers. Thus, global change will impact both nitrogen
fixation and carbon fixation. This dissertation is focused on describing interactions of
some of these environmental variables on N
2
fixation and CO
2
fixation by two species of
oceanic cyanobacteria, Trichodesmium erythraeum and Crocosphaera watsonii.
In this relatively short preface to the dissertation, I hope to have given a
somewhat simplistic view of the carbon and nitrogen cycles and how they are connected
through oceanic biological processes. I hope that you might seek more detailed
information about these processes with the references that I provided. As I mentioned,
we do not know with certainty how these feedback systems will respond to rapid global
change but we are certain that they are changing very quickly. The following chapters
describe some of the ways in which global change might impact the nitrogen and carbon
cycles.
xxvii
References in the Preface
Alley, R.B., Berntsen, T., Bindoff, N.L., Chen, Z. and others. Summary for
policymakers. In: Solomon S, Qin D, Manning M, Chen Z and others (Eds.) Climate
change 2007: The physical science basis. Contribution of Working Group I to the fourth
assessment report of the Intergovernmental Panel on Climate Change. Cambridge
University Press, Cambridge and New York. (2007).
Barcelos e Ramos, J., Biswas, H., Schulz, K. G., LaRoche, J. & Riebesell, U. 2007.
Effect of rising atmospheric carbon dioxide on the marine nitrogen fixer Trichodesmium.
Glob. Biogeochem. Cycles 21:GB2028.
Broecker, W.S. 1987. The biggest chill. Nat. Hist. Mag. 97:74-82.
Caldeira, K. & Wickett, M.E. 2003. Anthropogenic carbon and ocean pH. Nature.
425:365.
Capone, D. G., Zehr, J. P., Paerl, H. W., Bergman, B. & Carpenter, E. J. 1997.
Trichodesmium, a globally significant marine cyanobacterium. Science 276:1221-1229.
Capone, D.G. 2000. The marine microbial nitrogen cycle. In: Kirchman, D.L. (ed.)
Microbial ecology of the oceans. JohnWiley & Sons, Oxford, U.K., pp. 455-493.
Capone, D.G. & Knapp, A.N. 2007. A marine nitrogen cycle fix? Nature 115:159-160.
Falcon, L.I., Carpenter, E.J., Cipriano, F., Berman, B., & Capone, D.G. 2004. N
2
fixation
by unicellular bacterioplankton from the Atlantic and Pacific Oceans: Phylogeny and In
Situ Rates. Appl. Environ. Microbiol. 70:765-770.
Fu, F.-X., Mulholland, M. R., Garcia, N. S., Beck, A., Bernhardt, P. W., Warner, M. E.,
Sanudo-Wilhelmy, S. A. & Hutchins, D. A. 2008. Interactions between changing pCO
2
,
N
2
fixation, and Fe limitation in the marine unicellular cyanobacterium Crocosphaera.
Limnol. Oceanogr. 53:2472-2484.
Feely, R.A., Sabine, C.L., Hernandez-Ayon, J.M., Ianson, D. & Hales, B. 2008. Evidence
for upwelling of corrosive “acidified” water onto the continental shelf. Science.
320:1490-1492.
Gruber, N. 2008. The marine nitrogen cycle: overview and challenges. pp. 1-50. In:
Nitrogen in the Marine Environment, 2
nd
edition. D. G. Capone, D. A. Bronk, M. R.
Mulholland and E. J. Carpenter [Eds.], Elsevier Press, Amsterdam.
Hutchins, D. A., Fu, F. X., Zhang, Y., Warner, M. E., Feng, Y., Portune, K., Bernhardt,
P. W. & Mulholland, M. R. 2007. CO
2
control of Trichodesmium N
2
-fixation,
photosynthesis, growth rates, and elemental ratios: Implications for past, present, and
future ocean biogeochemistry. Limnol. Oceanogr. 52:1293-1304.
xxviii
Imbrie, J., Boyle, E. A., Clemens, S. C., Duffy, A., Howard, W. R., Kukla, G., Kutzbach,
J., Martinson, D. G., Mclntyre, A., Mix, A. C., Molfino, B., Morley, J. J., Peterson, L. C.,
Pisias, N. G., Prell, W. L., Raymo, M. E., Shackletons, N.J., & Toggweiler, J. R. 1992.
On the structure and origin of major glaciation cycles 1. Linear responses to
Milankovitch forcing. Paleocean.7:701-738.
Levitan, O., Rosenberg, G., Šetlík, I., Setlikova, E., Grigel, J., Klepetar, J., Prasil, O. &
Berman-Frank, I. 2007. Elevated CO
2
enhances nitrogen fixation and growth in the
marine cyanobacterium Trichodesmium. Glob. Change Biol. 13:531-538.
Kamykowski, D. & Zentara, S.J. 1990. Hypoxia in the world ocean as recorded in the
historical data set. Deep-Sea Res. 37:1861-1874.
Kuypers, M. M. M., Sliekers, A.O., Lavik, G., Schmid, M., Jørgensen, B.B.,
Kuenen, J.G., Sinninghe, J.S., Damsté, M., Strous, M. & Jetten, M.S.M. 2003. Anaerobic
ammonium oxidation by anammox bacteria in the Black Sea. Nature. 422, 608–611.
Kuypers, M. M., Lavik, G., Woebken, D., Schmid, M., Fuchs, B.M., Amann, R., B.B.
Jørgensen, & M.S.M. Jetten. 2005. Massive nitrogen loss from the Benguela upwelling
system through anaerobic ammonium oxidation. PNAS. 102: 6478–6483.
Orr, J.C, Fabry, V.J., Aumont, O., Bopp, L., Doney, S.C., Feely, R.A., Gnanadesikan6,
A., Gruber, N., Ishida, A., Joos, F., Key, R.M., Lindsay, K., Maier-Reimer, E., Matear,
R., Monfray, P., Mouchet, A., Najjar, R.G., Plattner, G.K., Rodgers, K.B., Sabine, C.L.,
Sarmiento, J.L., Schlitzer, R., Slater, R.D., Totterdell, I.J., Weirig, M.-F., Yamanaka, Y.,
& Yool, A. 2007. Anthropogenic ocean acidification over the twenty-first century and its
impacts on calcifying organisms. Nature 437:681-686.
Petit, J.R., Jouzel, J., Raynaud, D., Barkov, N.I., Barnola, J.-M., Basile, I., Bender, M.,
Chapellaz, J., Davis, M., Delaygue, G., Delmott, M., Kotlyakov, V.M., Legrand, M.,
Lipenkov, V.Y., Lorius, C., Pépin, L., Ritz, C., Saltzman, E. & Stievenard, M. 1999.
Climate and atmospheric history of the past 420,000 years from the Vostok ice core,
Antarctica. Nature 399:429-436.
Raven, J., Caldeira, K., Elderfield, H., Hoegh-Guldberg, O., Liss, P., Riebesell, U.,
Shepherd, H., Turley, C., & Watson, A. 2005. Ocean acidification due to increasing
atmospheric carbon dioxide. The Royal Society. Clyveden Press Ltd. Cardiff, UK
Redfield, A. C., B. H. Ketchum, and F. A. Richards, The influence of organisms on the
composition of sea-water, in The Sea, edited by M. N. Hill, vol. 2, pp. 26–77, Wiley-
Interscience, New York, 1963.
Riebesell, U. 2004. Effects of CO
2
enrichment on marine phytoplantkton. J. Oceanogr.
60:719-729.
xxix
Stramma, L., Johnson, G. C., Sprintall, J. & Mohrholz, V. 2008. Expanding oxygen
minimum zones in the tropical oceans. Science. 320:655-658.
Zehr, J. P., Waterbury, J. B, Turner, P. J., Montoya, J. P., Omoregie, E., Steward, G. F.,
Hansen, A., & Karl, D. M. 2001. Unicellular cyanobacteria fix N
2
in the subtropical
North Pacific Ocean. Nature 412: 635-638.
Zehr, J. P., Bench, Carter, B. J., Hewson, I., Niazi, F., Shi, T., Tripp H. J., & Affourtit, J.
P. 2008. Globally distributed uncultivated oceanic N
2
-fixing cyanobacteria lack oxygenic
photosystem II. Science 322:1110-1112.
xxx
Figures referenced in the Preface
CO CO
2 2
Carbon in marine sediment Carbon in marine sediment
Magma Magma
Carbon Carbon
Volcano Volcano
CO CO
2 2
+ + photosynth photosynth. .
organic carbon organic carbon
Atmospheric CO Atmospheric CO
2 2
Sea floor Sea floor
Ca Ca
+2 +2
+ 2HCO + 2HCO
3 3
- -
CaCO CaCO
3 3
+ H + H
2 2
O + CO O + CO
2 2
HCO HCO
3 3
- -
HCO HCO
3 3
- -
CO CO
2 2
+ H + H
2 2
O O H H
2 2
CO CO
3 3
HCO HCO
3 3
- -
+ H + H
+ +
CO CO
3 3
2 2- -
+ H + H
+ +
carbonic acid carbonic acid bicarbonate bicarbonate carbonate carbonate
* * * *
Accretionary Accretionary prism prism
of marine sediment of marine sediment
Respiration Respiration
Tectonic plate movement Tectonic plate movement
Subduction Subduction
zone zone
Ocean surface Ocean surface
calcium carbonate calcium carbonate
CaCO CaCO
3 3
HCO HCO
3 3
- -
Combustion Combustion
sediments sediments
Euphotic Euphotic zone zone
200 200
4000 4000
2000 2000
3000 3000
Depth from surface (m) Depth from surface (m)
1000 1000
Preface Figure 1. The oceanic carbon cycle. The oceans absorb atmospheric carbon
dioxide (CO
2
), which reacts with water to form carbonic acid (H
2
CO
3
) and immediately
dissociates into bicarbonate (HCO
3
-
) and a hydrogen ion (H
+
). Bicarbonate further
dissociates into carbonate (CO
3
2-
) and another H
+
. *The oceans become acidified as
increasing atmospheric CO
2
concentrations dissolve into the water and increase the H
+
concentration and decrease the pH. As a result of increasing atmospheric CO
2
concentrations, phytoplankton will likely increase their use of CO
2
during photosynthesis,
thereby increasing primary production rates in the euphotic zone (upper 200 meters of the
water column). The majority of these organic carbon molecules, however, are respired
back to CO
2
as they sink. Bicarbonate is the largest inorganic carbon reserve in the
ocean. Certain organisms (e.g. the phytoplanktonic coccolithophore Emiliana huxleyi)
create calcium carbonate (CaCO
3
) shells using two molecules of HCO
3
-
to form one
molecule of CaCO
3
. In this process, one molecule of CO
2
is released back to the
atmosphere. These CaCO
3
shells eventually sink and accumulate on the sea floor in
marine sediments, as they cannot be respired. Formation and sinking of shells are a major
process that removes dissolved inorganic carbon from the ocean and ultimately from the
atmosphere. The CaCO
3
-
is eventually combusted by heat from the earth’s mantle as
tectonic plates are subducted and CO
2
is blown back to the atmosphere out of volcanoes.
Preface Figure 1
xxxi
Preface Figure 2. The Great Ocean Conveyor Belt (from Broecker 1987). Oceanic surface
water flows north in the Atlantic Ocean. Surface water is heated in the tropics and
subtropics and evaporates in colder climates as it moves into higher latitudes in the
Northern Hemisphere. The dense, cold and more saline surface water sinks near Iceland
and flows South in the interior of the ocean in “intermediate” water. Intermediate water
accumulates nutrients as dead organic matter continuously rains out of surface layers
from above. The interior intermediate water also becomes increasingly low in oxygen
content as organisms consume it during respiration of sinking organic matter. For this
same reason, the concentration of CO
2
also increases in intermediate water. Some of the
nutrient-rich intermediate water is upwelled in the Indian Ocean but the majority is
upwelled in the Pacific Ocean and the high nutrient concentration supports high primary
production rates in the euphotic zone. This cycle is completed every ~1000 years.
Preface Figure 2
xxxii
Preface Figure 3. Concentrations of atmospheric CO
2
over the past 420,000 years as
recorded from air bubbles trapped ice cores above Lake Vostok, Antarctica (modified
from Petit et al. 1999). The partial pressure of CO
2
(pCO
2
) in the atmosphere consistently
cycled every ~100,000 years from approximately 180-190 parts per million by volume
(p.p.m.v.) during glacial periods to 280-300 p.p.m.v. during interglacial periods. The
present-day atmospheric pCO
2
concentration is ~386 p.p.m.v. and is predicted to be
between 750-1000 p.p.m.v within the next 100 years.
Preface Figure 3
xxxiii
OrgN
Glutamate
NH
4
+
Ammonium
NH
3
Ammonia
N
2
O
Nitrous oxide
NO
Nitric oxide
-3 -2 -1 0 +1 +2 +3 +4 +5
NO
3
-
Nitrate
NO
2
-
Nitrite
N
2
Dinitrogen
Oxidation state
Nitrite reductase
Monooxygenase (Fe, Cu)
Anammox
Hydrazine hydrolase
Nitrite oxidoreductase
Nitrate reductase (Fe)
Nitrogen fixation
Denitrification
Nitrification
Nitrogenase (Fe, Mo)
Nitrous oxide
reductase
Nitric oxide
reductase
Nitrite reductase
(Gas)
(Gas)
(Gas)
Assimilation
Ammonification
Preface Figure 4. The marine nitrogen cycle (modified from Capone 2000) with chemical
species of inorganic nitrogen and their respective oxidation states. The largest reservoir
of nitrogen exists as dinitrogen (N
2
) gas in the atmosphere and can be reduced to
ammonium (NH
4
+
) by N
2
-fixing organisms with the enzyme nitrogenase that contains
iron and molybdenum as metal cofactors. Ammonium can then be assimilated into
glutamate, an amino acid and other organic molecules that contain N (OrgN).
Ammonium is oxidized to ammonia (NH
3
) and can then be converted to nitrite (NO
2
-
) by
oxidizing prokaryotes that contain monooxygenase with iron and copper cofactors. In the
reverse reaction NO
2
-
is reduced to ammonia by nitrite reductase by other groups of
prokaryotes. Nitrite is oxidized to nitrate (NO
3
-
) by nitrite oxidoreductase. The enzyme
nitrate reductase reduces NO
3
-
to NO
2
-
, which is then reduced to gaseous nitric oxide
(NO) with nitrite reductase. The enzyme nitric oxide reductase reduces NO to gaseous
nitrous oxide (N
2
O) which is then reduced to N
2
with nitrous oxide reductase. In a
relatively recently discovered pathway termed anammox, NH
4
+
is oxidized as NO
2
-
is
reduced to form N
2
with the enzyme hydrazine hydrolase.
Preface Figure 4
xxxiv
1000
Depth from
sea surface (m)
100
500
30 20 10 0
[NO
3
-
] (µM)
Preface Figure 5. Nitrate concentrations as a function of depth from the ocean surface. In
surface layers, light drives primary production of photosynthetic organisms that take up
and reduce nitrate (NO3-) to ammonium before assimilating it into organic nitrogen
compounds. Nitrate concentrations are highest in intermediate waters below 1000 meters
from the surface. These intermediate waters are eventually upwelled and supply primary
producers with high concentrations of macronutrients such as nitrate and phosphate.
High primary production rates in these upwelling regions create oxygen minimum zones
in underlying water.
Preface Figure 5
xxxv
Preface Figure 6. Regions of major oxygen minimum zones (OMZ’s) in the world’s
oceans (shaded areas represent OMZ’s; from Kamykowski and Zentara 1990). High
concentrations of macronutrients such as nitrate and phosphate are upwelled from
intermediate waters in these regions and support high primary production rates.
Decaying organic material that results from these high primary production rates sinks and
is respired at depth. Oxygen minimum zones are created by the consumption of organic
carbon and oxygen (O
2
). Nitrate (NO
3
-
) serves as an alternative electron acceptor for
further respiration of organic carbon, when O
2
is depleted. In this process termed
denitrification, NO
3
-
is reduced to dinitrogen gas. Alternatively, the anammox reaction
reduces NO
3
-
and ammonium to N
2
gas in oxygen minimum zones.
Preface Figure 6
xxxvi
Contributions of Authors
In chapter one, I performed the majority of culturing, designing of experiments
and sampling of cultures of Trichodesmium erythraeum. I analyzed the data and wrote the
paper with guidance and comments from David Hutchins and anonymous reviewers. Fei-
Xue Fu helped with some of the culturing and sampling of Trichodesmium erythraeum.
Cynthia L. Breene helped with sampling of cultures of Trichodesmium erythraeum. Peter
W. Bernhardt analyzed samples for the calculation of
15
N
2
fixation rates. Margaret R.
Mulholland helped with analysis and calculation of
15
N
2
fixation rates. Jill A. Sohm
performed some of the gross N
2
fixation assays and calculated some of the gross N
2
fixation rates. David A. Hutchins helped with some of the composition and direction of
the paper as well as guidance with respect to experimental designs.
In chapter two, I performed the majority of culturing, designing of experiments
and sampling of cultures of Crocosphaera watsonii. I analyzed the data and wrote the
paper with guidance and comments from David Hutchins and anonymous reviewers. Fei-
Xue Fu helped with some of the culturing and sampling of Crocosphaera watsonii.
Elizabeth K. Yu helped with sampling of cultures of Crocosphaera watsonii. Cynthia L.
Breene helped with sampling of cultures of Crocosphaera watsonii. Peter W. Bernhardt
analyzed samples for the calculation of
15
N
2
fixation rates. Margaret R. Mulholland
helped with analysis and calculation of
15
N
2
fixation rates. David A. Hutchins helped
with some of the composition and direction of the paper as well as guidance with respect
to experimental designs.
xxxvii
In chapter three, I performed the majority of culturing, designing of experiments
and sampling of cultures of Crocosphaera watsonii. I also analyzed the data and wrote
the paper with guidance and comments from David Hutchins. Fei-Xue Fu helped with
some of the culturing and sampling of Crocosphaera watsonii. Elizabeth K. Yu helped
with sampling of cultures of Crocosphaera watsonii. David A. Hutchins helped with
some of the composition and direction of the paper as well as guidance with respect to
experimental designs.
1
Introduction
Our global climate is changing rapidly. Models predict that anthropogenic
burning of fossil fuels, deforestation, and production of concrete will cause the present-
day atmospheric concentration of carbon dioxide (CO
2
) of ~390 parts per million (ppm)
to double within the next 100 years (Raven et al. 2005, Alley et al. 2007-IPCC). Since
the dawn of the industrial revolution, atmospheric pCO
2
has increased by ~35% from pre-
industrial concentrations of ~280 ppm. Carbon dioxide production has accelerated
exponentially to rates that may be unprecedented in the history of the earth. Before the
present interglacial period, atmospheric pCO
2
had consistently cycled between ~190 and
280 ppm during glacial and interglacial periods, respectively, for the past 420,000 years
(Petit et al. 1999). Projected 100-year estimates of atmospheric pCO
2
range between
750-1000 ppm and exceed interglacial levels by approximately 170-260%. Due to effects
of the predicted rise in atmospheric CO
2
on the seawater carbonate buffer system, the pH
of the world’s oceans is expected to decrease by approximately 0.35 units over the next
100 years (Caldeira and Wickett 2003). This is equivalent to a 125% increase in the
oceanic H
+
ion concentration, and will drastically change the ecosystems and
biogeochemistry of the oceans. This process is called ocean acidification.
This abrupt rise in the atmospheric concentration of CO
2
is an extremely
challenging issue. The only long-term solution to this problem is to end human reliance
on burning fossil fuels for energy generation. From a scientific perspective, it is also
imperative to understand how C circulates through global systems and how this will
change in the future. Most of the world’s C is locked in sedimentary rocks (Raven et al.
2
2005; Bianchi 2011). The oceans absorb atmospheric CO
2
, which is “fixed” by algae
during photosynthesis and calcification and then transferred to the deep ocean by the
biological pump to be stored in deep water sediments for thousands of years or buried in
sedimentary rocks for millions of years (Bianchi 2011). Thus, the oceans have a
tremendous capacity to sequester atmospheric CO
2
and store C. Through biological
processes like photosynthesis and the elemental ratios of phytoplankton, the C cycle is
inextricably linked with the nitrogen (N) cycle. Nitrogen is one of the major nutrients that
limit primary production rates in the world’s oceans (Capone 2008). Because of this, a
clear understanding of the oceanic N cycle is essential to a clear understanding of the C
cycle.
Deep water N reaches the ocean surface mainly by vertical advection and
diffusion and is considered “new” N because it leads to drawdown of atmospheric CO
2
by stimulating positive primary production rates and sinking of organic C (Falkowski et
al. 1998). However, deep-water N fluxes to surface waters are accompanied by
upwelling and diffusion of deep-water CO
2
, which reduces the impact of primary
production on drawdown of atmospheric CO
2
(Capone et al. 1997). Because atmospheric
inputs of N to the euphotic zone are not accompanied by inputs of CO
2
from deep water,
fixation of atmospheric N
2
has a much stronger impact on biological sequestration of
atmospheric CO
2
and carbon export. For this reason, oceanic N
2
fixers are key biological
components that link global N and C biogeochemical cycles. Within the next 100 years,
oceanic N
2
-fixation rates will likely increase thereby accelerating effects on oceanic
primary production rates and carbon export (Barcelos e Ramos et al. 2007; Hutchins et al.
3
2007; Levitan et al. 2007; Kranz et al. 2010; Fu et al. 2008; Hutchins et al. 2009; Garcia
et al. 2011).
Within the past two decades, oceanic N
2
fixation has received considerable
attention primarily because it had previously been grossly underestimated. Nevertheless,
current estimates of oceanic N
2
fixation are still lower than estimates of denitrification,
ranging between 100-200 Tg yr
-1
compared with estimates of 200-400 Tg yr
-1
for
denitrification (Capone 2008). This mismatch between N supply and removal processes
suggests that the N cycle could be out of balance, although this is unlikely because
theoretical and empirical evidences suggest that denitrification and N
2
-fixation rates are
closely coupled (Falkowski et al. 1997; Deutsch et al. 2007; Capone and Knapp 2007).
Instead, the difficulty of sampling N
2
-fixation rates accurately and our general lack of
knowledge with respect to the sources and ranges of oceanic N
2
fixation may responsible
for this gap in N
2
fixation and denitrification estimates (Moisander et al. 2010).
Historically, the majority of field and laboratory studies examining oceanic N
2
fixation have focused on filamentous N
2
fixers belonging to the genus Trichodesmium
(Capone et al. 1997; 2005). Trichodesmium dominates N-limited regions of the
subtropical and tropical oceans and is restricted to these regions because of its limited
temperature range for growth (> ~20
o
C, Breitbarth et al. 2007). Nitrogen fixation by the
5 recognized species of Trichodesmium might contribute approximately half of marine N
2
fixation, as they thrive in regions like the Sargasso Sea (Sanudo-Wilhelmy et al. 2001,
Capone et al. 2005), the Arabian Sea (Capone et al. 1998) and the North Pacific Ocean
(Karl et al. 1997).
4
Recently, unicellular oceanic diazotrophs have received more attention, after they
were recognized as also being substantial contributors to global oceanic N
2
fixation (Zehr
et al. 2001; Montoya et al. 2004; Zehr et al. 2007; 2008; Church et al. 2008). Zehr and
colleagues (2001; 2007) identified two types of oceanic unicellular N fixers with different
size ranges and genomic diversity. One uncultivated group, UCYN A does not possess
an O
2
-generating photosystem II, and thus has tremendous implications for the N and C
cycles because of its likely net positive contribution to the global reactive N pool and
likely net positive consumption of organic C (Zehr et al. 2008). Unicellular N
2
fixers are
thought to occupy larger regions of the oceans than Trichodesmium, due to their wide-
ranging growth capabilities with respect to temperature and irradiance (Moisander et al.
2010). Early estimates suggested that unicellular diazotrophs might contribute roughly
half of oceanic N
2
fixation (Zehr et al. 2001; Montoya et al. 2004; Church et al. 2008),
but as new discoveries are made, this estimate continues to rise and narrow the largely
unbalanced N budget (Deutsch et al. 2007; Moisander et al. 2010). Recent studies have
focused on characterizing the physiological and biochemical traits of Crocosphaera
watsonii, a species belonging to the group UCYN B, because it seemingly represents a
large portion of the unicellular community in terms of N
2
fixation (Fu et al. 2008; Mohr
et al. 2010; Garcia et al. 2013, in-press). Crocosphaera watsonii thus serves as a model
organism to understand how photosynthetic unicellular diazotrophs function in the
biogeochemical cycles of N and C (Zehr et al. 2001; Berman-Frank et al. 2007; Goebel et
al. 2008; Fu et al. 2008; Webb et al. 2009; Mohr et al. 2010; Saito et al. 2011; Garcia et
al. in press).
5
Because of the strong biogeochemical link between N
2
fixation and the C cycle,
many studies have focused on nutrient controls of oceanic N
2
-fixation rates. Some
studies suggest that iron (Fe) is the primary limiting nutrient of N
2
fixation in the Pacific
Ocean (Wu et al. 2000) and phosphorus (P) is more important in the central North
Atlantic Ocean (Sanudo-Wilhelmy et al. 2001). Some studies indicate that the North
Pacific Gyre may shift towards P-limited communities during El Niño events (Karl et al.
1995; 1997). More recently, studies suggest that Fe and P colimitation of N
2
-fixation
may be highly influential on oceanic diazotrophs (Garcia et al. in-preparation),
specifically in the eastern tropical Atlantic Ocean (Mills et al. 2004) and the
Mediterranean Sea (Ridame et al. 2011).
In the vast Pacific Ocean, atmospheric Fe deposition is much lower than in the
Atlantic Ocean and as a result, Fe limits primary production rates in the North Pacific
Gyre, eastern North Pacific Ocean and eastern equatorial Pacific region (Hutchins et al.
1998; DiTullio et al. 2005; Leblanc et al. 2005). Conversely, mineral dust deposition
rates in the tropical North Atlantic Ocean are among the highest in the world (Gao et al.
2001). Estimates of aeolian Fe input to the Sargasso Sea (0.2 - 0.8 µmol Fe m
-2
d
-1
) are
several times higher than those reported for the Pacific Ocean near Hawaii (0.08 – 0.16
µmol Fe m
-2
d
-1
, Wu et al. 2000). These elevated Fe deposition rates in the North Atlantic
Ocean are thought to be suitable to support high N
2
-fixation rates. For example, Falcon et
al. (2004) documented N
2
-fixation rates by unicellular cyanobacteria in the tropical North
Atlantic Ocean that were 1-2 orders of magnitude higher than rates in the subtropical
North Pacific Ocean. These data support the hypothesis presented by Wu et al. (2000)
6
suggesting that Fe is the major limiting nutrient in Pacific waters with respect to N
2
-
fixation rates, while P is limiting in the North Atlantic Basin.
Nitrogen-fixing cyanobacteria have cellular Fe quotas that are 5-10 times higher
than those of non-N
2
-fixing phytoplankton, mainly due to the high Fe demand of the N
2
-
fixing enzyme nitrogenase (Kustka et al. 2003). The nitrogenase complex has a
homodimeric Fe protein with a 4Fe:4S metallocluster, a heterotetrameric protein with an
8Fe:7S P cluster and a 7Fe:1Mo cofactor (Howard and Rees 1996). As a consequence of
this high biochemical Fe demand, Berman-Frank and colleagues (2007) documented high
Fe requirements for N
2
fixation and growth of laboratory cultures of Trichodesmium
erythraeum. Iron quotas and requirements for unicellular cyanobacteria like
Crocosphaera are still not well documented, although they may be considerably lower
than those of Trichodesmium (Berman-Frank et al. 2007; Fu et al. 2008; Saito et al.
2011).
With respect to P, all living organisms use it in the backbone structure for coding
deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), and most also use it in
cellular membranes as a component of phospholipids. Some species of phytoplankton
including Trichodesmium are known to replace phospholipids with sulfolipids under P-
limiting conditions, but this does not seem to be the case for Crocosphaera (Van Mooy et
al. 2006, 2009). In addition, phytoplankton cells require P for their energy currency:
ATP, ADP and cyclic AMP. With respect to P-acquisition, Trichdoesmium seems to
have an advantage over other phytoplankton species through their ability to scavenge P
from phosphonates, which represent a substantial portion (~25%) of the organic P pool in
7
the open ocean (Dyhrman et al. 2006). Crocosphaera does not appear to have this
capability, but does have genes that encode for high-affinity P transport and can access P
from other types of organic P molecules with a putative alkaline phosphatase (Dyhrman
& Haley 2006).
In general, the North Atlantic Ocean has been considered P-limited in comparison
with the Pacific Ocean. This surplus of P in the Pacific Ocean, known as +P*, is simply a
high ratio of P:N over the “normal” Redfield ratio of 1:16 (Redfield et al. 1963). The
excess P in the Pacific Ocean is caused by high denitrification and/or anammox rates in
oxygen (O
2
) minimum zones that underlie upwelling regions of the Pacific Ocean off the
coast of Peru. Such large O
2
minimum zones are not as prominent in the North Atlantic
Ocean due to the fact that deep water in the North Atlantic is new with respect to its
removal from atmospheric contact, and so contains relatively high concentrations of O
2
.
High denitrification rates also exist in the water columns of the Arabian Sea, off the west
coast of Africa, and the in sub-arctic region of the North Pacific Ocean, as well as in
shallow marine sediments worldwide. Deutsch et al. (2007) modeled N
2
fixation using
an ocean circulation model on the basis that a +P* will likely result in N
2
fixation. Their
data indicated that the highest rates of N
2
fixation occur in the Pacific Ocean where
denitrification rates are high, but atmospheric input of Fe is low. This finding suggests
that Fe might not be the principle factor in limiting N
2
-fixation rates in the world’s
oceans, and that P from deep water might be a more dominant controlling factor.
Alternatively, however, upwelled Fe from subsurface waters might augment the lower
atmospheric Fe source sufficiently to supply enough Fe for N
2
fixation. Overall, their
8
model supports the general idea that reactive N input by N
2
fixation balances oceanic
reactive N removal by denitrification. In this regard, the controversy over Fe and P
limitation of N
2
fixation is subject to further debate, and examination of the colimitation
interactions between these two key controlling nutrients is paramount.
Major input rates of Fe and P to surface layers of the ocean are likely to change
over the next 100 years. Phosphorus input to the surface of the open ocean is mostly
attributed to upwelling and diffusion from deep water and to fluvial sources, while the
major input of Fe occurs by aeolian deposition (Falkowski et al. 1998; Mills et al. 2004).
As a result of increasing global temperatures, surface layers of the ocean will become
more stratified, thereby decreasing P input to surface layers from deep water (Sarmiento
et al. 2004; Hutchins et al. 2009). Iron input from continental dust to the ocean surface is
also likely to change as the global climate changes, although the direction of this shift is
unclear (Michaels et al. 2001, Mahowald et al. 2006, Woodward et al. 2005). For
instance, a warmer climate may create drier continents or conversely increase
precipitation rates, thereby changing dust deposition rates to the oceans. Atmospheric
dust deposition is a minor source of P to the ocean surface, and it is generally uncoupled
from Fe fluxes because of their different solubility constants and dependencies on
precipitation and atmospheric moisture content (Ridame and Guieu 2002). Because the
major inputs of P and Fe to the surface layers of the oceans occur by different
mechanisms, these input rates are uncoupled and will likely change disproportionately in
space and time over the next 100 years.
9
Along with these historically recognized controlling factors and its high total
concentration in surface seawater, CO
2
has only recently been scrutinized as a critical
limiting nutrient for growth and N
2
fixation by oceanic diazotrophs. Several studies have
documented increased in N
2
-fixation rates by T. erythraeum and C. watsonii in response
to elevated pCO
2
(Hutchins et al. 2007; Barcelos e Ramos et al. 2007; Levitan et al. 2007;
Fu et al. 2008; Kranz et al. 2009; Kranz et al. 2010; Levitan et al. 2010; Garcia et al.
2011; Garcia et al. in press). Data reported by Hutchins et al. (2007) suggested that P and
CO
2
effects on N
2
-fixation rates by T. erythraeum were independent of each other,
because elevated pCO
2
stimulated N
2
fixation in a manner that was proportionately nearly
identical in P-limited and P-replete laboratory cultures. Collectively, these findings
suggest that elevated pCO
2
will probably have a positive effect on N
2
-fixation rates in the
future ocean. Some estimates suggested that global N
2
-fixation rates by Trichodesmium
might increase by 50-60%, if the responses of laboratory cultures are similar to those of
diazotrophs in the field. Recent experiments, however, indicated that the effect of
elevated pCO
2
is probably more important at low-light intensities (Kranz et al. 2010,
Levitan et al. 2010, Garcia et al. 2011). Because the majority of Trichodesmium N
2
fixation occurs near the surface due to buoyant colonies (Capone et al. 1998), the water-
column-integrated effect might be smaller, specifically because phytoplankton will
experience brighter light densities in a future, more stratified water column. Thus, field
experiments are needed that examine responses of elevated pCO
2
in combination with
changing light, Fe and P.
10
We know that environmental factors such as biologically required element
concentrations (e.g. CO
2
, Fe, and P), light, and temperature are going to change in the
ocean surface within the next 100 years. In order to accurately predict how our rapidly
changing climate will impact the global N and C cycles, we need to understand how these
environmental factors will interact with each other to affect marine N
2
-fixation rates. The
results presented here thus focus on an examination of a wide variety of mutual
interactions between light, P and CO
2
as they influence N
2
-fixation rates by multiple
strains of T. erythraeum and C. watsonii.
11
References in the Introduction
Alley, R.B., Berntsen, T., Bindoff, N.L., Chen, Z. and others. Summary for
policymakers. In: Solomon S, Qin D, Manning M, Chen Z and others (Eds.) Climate
change 2007: The physical science basis. Contribution of Working Group I to the fourth
assessment report of the Intergovernmental Panel on Climate Change. Cambridge
University Press, Cambridge and New York. (2007).
Barcelos e Ramos, J., Biswas, H., Schulz, K. G., LaRoche, J. & Riebesell, U. 2007.
Effect of rising atmospheric carbon dioxide on the marine nitrogen fixer Trichodesmium.
Glob. Biogeochem. Cycles 21:GB2028.
Berman-Frank, I., Quigg, A., Finkel, Z. V., Irwin, A. J. & Haramaty, L. 2007. Nitrogen-
fixation strategies and Fe requirements in cyanobacteria. Limnol. Oceanogr. 52:2260–
2269.
Bianchi, T.S. 2011. The role of terrestrially derived organic carbon in the coastal ocean:
A changing paradigm and the priming effect. PNAS. 108:19473-19481.
Breitbarth, E., Oschlies, A., & LaRoche J. 2007. Physiological constraints on the global
distribution of Trichodesmium - effect of temperature on diazotrophy. Biogeosciences.
4:53-61.
Caldeira, K. & Wickett, M.E. 2003. Anthropogenic carbon and ocean pH. Nature.
425:365.
Capone, D. G., Subramanium, A., Montoya, J. P., Voss, M., Humborg, C., Johansen,
A.M., Siefert, R. L. & Carpenter, E. J. 1998. An extensive bloom of the N
2
-fixing
cyanobacterium Trichodesmium erythraeum in the central Arabian Sea. Mar. Ecol. Prog.
Ser. 172:281-292.
Capone, D. G., Zehr, J. P., Paerl, H. W., Bergman, B. & Carpenter, E. J. 1997.
Trichodesmium, a globally significant marine cyanobacterium. Science 276:1221-1229.
Capone, D. G., Burns, J. A., Montoya, J. P., Subramaniam, A., Mahaffey, C., Gunderson,
T., Michaels, A. F. & Carpenter, E. J. 2005. Nitrogen fixation by Trichodesmium spp.:
An important source of nitrogen to the tropical and subtropical North Atlantic Ocean.
Global Biogeochem. Cy. 19:GB2024.
Capone, D. G. 2008. The marine nitrogen cycle. Microbe 3:186-192.
Church, M. J., Björkman, K. M., Karl, D. M., Saito, M. A. & Zehr, J. P. 2008. Regional
distributions of nitrogen-fixing bacteria in the Pacific Ocean. 53:63-77.
12
Deutsch, C., Sarmiento, J. L., Sigman, D. M., Gruber, N. & Dunne, J. P. 2007. Spatial
coupling of nitrogen inputs and losses in the ocean. Nature 445:163-167.
Dyhrman, S. T. & Haley, S.T. 2006. Phosphorus Scavenging in the Unicellular Marine
Diazotroph Crocosphaera watsonii. App. Environ. Microb. 72:1452-1458.
Dyhrman S. T., Chappell, P. D., Haley, S. T., Moffett, J. W., Orchard, E. D., Waterbury,
J. B. & Webb, E. A. 2006. Phosphonate utilization by the globally important marine
diazotroph Trichodesmium. Nature 439:1452-1458.
Falkowski, P. G. 1997. Evolution of the nitrogen cycle and its influence on the biological
sequestration of CO
2
in the ocean. Nature 387:272-275.
Falkowski, P.G., Barber, R.T. & Smetacek, V. 1998. Biogeochemical controls and
feedbacks on ocean primary production. Science. 281:200-206.
Falcon, L.I., Carpenter, E.J., Cipriano, F., Berman, B., & Capone, D.G. 2004. N
2
fixation
by unicellular bacterioplankton from the Atlantic and Pacific Oceans: Phylogeny and In
Situ Rates. Appl. Environ. Microbiol. 70:765-770.
Fu, F.-X., Mulholland, M. R., Garcia, N. S., Beck, A., Bernhardt, P. W., Warner, M. E.,
Sanudo-Wilhelmy, S. A. & Hutchins, D. A. 2008. Interactions between changing pCO
2
,
N
2
fixation, and Fe limitation in the marine unicellular cyanobacterium Crocosphaera.
Limnol. Oceanogr. 53:2472-2484.
Gao, Y., Kaufman, Y.J., Tanre, D., Kolber, D., & Falkowski, P.G. 2001. Seasonal
distributions of Aeolian iron fluxes to the global ocean. Geophys. Res. Lett. 28:29-32
Garcia, N. S., Fu, F-X, Breene, C. L., Bernhardt, P. W., Mulholland, M. R. Sohm, J. A. &
Hutchins, D. A. 2011. Interactive effects of irradiance and CO
2
on CO
2
- and N
2
fixation
in the diazotroph Trichodesmium erythraeum (Cyanobacteria). J. Phycol. 47:1292-1303.
Garcia, N.S., Fu, F-X., Yu, E.K., Breene, C.L., Bernhardt, P.W., Mulholland, M.R. &
Hutchins, D.A. 2013. Combined effects of CO
2
and light on the unicellular N
2
-fixing
cyanobacterium Crocosphaera watsonii: A comparison of two isolates from the western
tropical Atlantic Ocean. Eur. J. Phycol. In press.
Goebel, N. L., Edwards, C. A., Carter, B. J., Achilles, K. M., & Zehr, J. P. 2008. Growth
and carbon content of three different-sized diazotrophic cyanobacteria observed in the
subtropical North Pacific. J. Phycol. 44:1212-1220.
Howard, J.B. & Reese, D.C. 1996. Structural basis of biological nitrogen fixation. Chem.
Rev. 96:2965-2982.
13
Hutchins, D.A., DiTullio, G.R., Zhang, Y. & Bruland, K.W. 1998. An iron limitation
mosaic in the California upwelling regime. Limnol. Oceanogr. 43:1037-1054
Hutchins, D. A., Fu, F. X., Zhang, Y., Warner, M. E., Feng, Y., Portune, K., Bernhardt,
P. W. & Mulholland, M. R. 2007. CO
2
control of Trichodesmium N
2
-fixation,
photosynthesis, growth rates, and elemental ratios: Implications for past, present, and
future ocean biogeochemistry. Limnol. Oceanogr. 52:1293-1304.
Hutchins, D. A., Mulholland, M. R., & Fu, F.-X. 2009. Nutrient cycles and marine
microbes in a CO
2
enriched ocean. Oceanography 22:128-145.
Karl, D.M., Letelier, R., Hebel, D., Tupas, L., Dore, J., Christian, J. & Winn, C. 1995.
Ecosystem changes in the North Pacific subtropical gyre attributed to the 1991-92 El
Niño. Nature. 373:230-234.
Karl, D., Letelier, R., Tupas, L., Dore, J., Christian, J. & Hebel, D. 1997. The role of
nitrogen fixation in biogeochemical cycling in the subtropical North Pacific Ocean.
Nature 388:533-538.
Kranz, S. A., Sultemeyer, D., Richter, K. U., & Rost, B. 2009. Carbon acquisition by
Trichodesmium: The effect of pCO
2
and diurnal changes. Limnol. Oceanogr. 54:548-559.
Kranz, S. A., Levitan, O., Richter, K-U., Prášil, O., Berman-Frank, I., Rost, B. 2010.
Combined effects of CO
2
and light on the N
2
-fixing cyanobacterium Trichodesmium
IMS101: Physiological responses. Plant Physiol. 154:334-345.
Kustka, A. Sanudo-Wilhelmy, S., Carpenter, E. J., Capone, D. G. & Raven, J. A. 2003a.
A revised estimate of the iron use efficiencies of nitrogen fixation with special reference
to the marine cyanobacterium Trichodesmium spp. (Cyanophyta). J. Phycol. 39:12-35.
Leblanc, K., Hare, C.E., Boyd, P.W., Bruland, K.W., Sohst, B., Pickmere, S., Lohan,
M.C., Buck, K., Ellwood, M. & Hutchins, D.A. 2005. Deep Sea Res. I. 52:1842-1864.
Levitan, O., Rosenberg, G., Šetlík, I., Setlikova, E., Grigel, J., Klepetar, J., Prasil, O. &
Berman-Frank, I. 2007. Elevated CO
2
enhances nitrogen fixation and growth in the
marine cyanobacterium Trichodesmium. Glob. Change Biol. 13:531-538.
Levitan, O., Kranz, S. A., Spungin, D., Prášil, O., Rost, B. & Berman-Frank, I. 2010.
Combined effects of CO
2
and light on the N
2
-fixing cyanobacterium Trichodesmium
IMS101: A mechanistic view. Plant Physiol. 154:346-356.
Mahowald, N.M., Yoshioka, M., Collins, W.D., Conley, A.J., Fillmore, D.W. &
Coleman, D.B. 2006. Climate response and radiative forcing from mineral aerosols
14
during the last glacial maximum, pre-industrial, current and doubled-carbon dioxide
climates. Geophys. Res. Lett. 33:L20705. DOI: 10.1029/2006GL026126.
Michaels, A. F., Karl, D. M., & Capone, D. G. 2001. Element stoichiometry, new
production and nitrogen fixation. 14:68-77.
Mills, M. M., Ridame, C., Davey, M., La Roche, J. and Geider, R. J. 2004. Iron and
phosphorus co-limit nitrogen fixation in the eastern tropical North Atlantic. Nature
429:292-294.
Mohr, W., Intermaggio, M.P. & J. LaRoche. 2010. Diel rhythm of nitrogen and carbon
metabolism in the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii
WH8501. Environ. Microbiol. 12:412-421.
Moisander, P. H., Beinart, R. A., Hewson, I., White, A. E., Johnson, K. S., Carlson, C.
A., Montoya, J. P, & Zehr, J. P. 2010. Fixation domain unicellular cyanobacterial
distributions broaden the oceanic N
2
fixation domain. Science 327:1512-1514.
Montoya, J. P., Holl, C. M., Zehr J. P., Hansen, A., Villareal, T. A. & Capone, D. G.
2004. High rates of N
2
fixation by unicellular diazotrophs in the oligotrophic Pacific
Ocean. 430:1027-1031.
Petit, J.R., Jouzel, J., Raynaud, D., Barkov, N.I., Barnola, J.-M., Basile, I., Bender, M.,
Chapellaz, J., Davis, M., Delaygue, G., Delmott, M., Kotlyakov, V.M., Legrand, M.,
Lipenkov, V.Y., Lorius, C., Pépin, L., Ritz, C., Saltzman, E. & Stievenard, M. 1999.
Climate and atmospheric history of the past 420,000 years from the Vostok ice core,
Antarctica. Nature 399:429-436.
Raven, J., Caldeira, K., Elderfield, H., Hoegh-Guldberg, O., Liss, P., Riebesell, U.,
Shepherd, H., Turley, C., & Watson, A. 2005. Ocean acidification due to increasing
atmospheric carbon dioxide. The Royal Society. Clyveden Press Ltd. Cardiff, UK
Redfield, A. C., B. H. Ketchum, and F. A. Richards, The influence of organisms on the
composition of sea-water, in The Sea, edited by M. N. Hill, vol. 2, pp. 26–77, Wiley-
Interscience, New York, 1963.
Ridame, C. & Guieu, C. 2002. Saharan input of phosphate to the oligotrophic water of the
open western Mediterranean Sea. Limnol. Oceanogr. 47:856-869.
Ridame, C., Le Moal, M., Guieu, C., Ternon, E., Biegala, I.C., L’Helguen., S. & Pujo-
Pay, M. 2011. Nutrient control of N
2
fixation in the oligotrophic Mediterranean Sea and
the impact of Saharan dust events. Biogeosci. 8:2773-2783.
15
Saito, M.A., Bertrand, E.M., Dutkiewics, S., Bulygen, V.V., Moran, D.M., Montiero,
F.M., Follows, M.J., Valois, F.W. & J.B. Waterbury. 2011. Iron conservation by
reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii.
Proc. Natl. Acad. Sci. 108:2184-2189.
Sañudo-Wilhelmy, S. A., Kustka, A. B., Gobler, C. J., Hutchins, D. A., Yang, M., Lwiza,
K., Burns, J., Capone, D. G., Raven, J. A. & Carpenter, E. J. 2001. Phosphorus limitation
of nitrogen fixation by Trichodesmium in the central Atlantic Ocean. Nature 411: 66-69.
Sarmiento, J. L., Slater, R., Barber, R., Bopp, L., Doney, S. C., Hirst, A. C., Kleypas, J.,
Matear, R., Mikolajewicz, U., Monfray, P., Soldatov, V., Spall, S. A. & Stouffer, R.
2004. Response of ocean ecosystems to climate warming. Global Biogeochem. Cy.
18:GB3003. doi:10.1029/2003GB002134.
Van Mooy, B.A.S., Rocap, G. Fredericks, H.F., Evans, C.T., Devol, A.H. 2006.
Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in
oligotrophic marine environments. PNAS. 103: 8607-8612.
Van Mooy, B.S., Fredericks, H.F., Pedler, B.E. Dyhrman, S.T., Karl, D.M., Koblížek, M.,
Lomas, M.W., Mincer, T.J., Moore, L.R., Moutin, T., Rappé, M.S., Webb, E.A. 2009.
Phytoplankton in the ocean use non-phospholipids in response to phosphorus scarcity.
Nature. 458: 69-72.
Webb, E.A., Ehrenreich, I.M., Brown, S.L., Valois, F.W. & J. B. Waterbury. 2009.
Phenotypic and genotypic characterization of multiple strains of the diazotrophic
cyanobacterium Crocosphaera watsonii, isolated from the open ocean. Environmental
Microbiology. 11-338-348.
Woodward, S., Roberts, D., & Betts, R. 2005. A simulation of the effect of climate
change induced desertification on mineral dust aerosol. Geophys. Res. Lett. 32:L18810.
doi:10.1029/2005GL023482.
Wu, J. F., Sunda, W., Boyle, E. A. & Karl, D. M. 2000. Phosphate depletion in the
western North Atlantic Ocean. Science 289:759-762.
Zehr, J. P., Bench, S. R., Mondragon, E. A., McCarren, J., & DeLong, E. F. 2007. Low
genomic diversity in tropical oceanic N
2
-fixing cyanobacteria. Proc. Nat. Acad. Sci.
1780-17812.
Zehr, J.P., Bench, S.R., Carter, B.J., Hewson, I., Niazi, F., Shi, T., Tripp, J. & Affourtit,
J.P. 2008. Globally distributed uncultivated oceanic N
2
-fixing cyanobacteria lack
oxygenic photosystem II. Science 322:1110-1112.
16
Chapter 1
INTERACTIVE EFFECTS OF IRRADIANCE AND CO
2
ON CO
2
- AND N
2
FIXATION IN THE DIAZOTROPH TRICHODESMIUM ERYTHRAEUM
(CYANOBACTERIA)
Manuscript published in Journal of Phycology, 2011
Nathan S. Garcia
Department of Biological Sciences, University of Southern California, Los Angeles, CA
90089, USA
Fei-Xue Fu
Department of Biological Sciences, University of Southern California, Los Angeles, CA
90089, USA
Cynthia L. Breene
Department of Biological Sciences, University of Southern California, Los Angeles, CA
90089, USA
Peter W. Bernhardt
Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University,
Norfolk, VA 23529, USA
Margaret R. Mulholland
Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University,
Norfolk, VA 23529, USA
17
Jill A. Sohm
Department of Biological Sciences, University of Southern California, Los Angeles, CA
90089, USA
David A. Hutchins
1
Department of Biological Sciences, University of Southern California, Los Angeles, CA
90089, USA
1
Corresponding author; dahutch@usc.edu, ph: (213) 740-5616
18
Running title: Light & CO
2
effects on Trichodesmium N
2
fixation
19
Chapter 1 Abstract
The diazotrophic cyanobacteria Trichodesmium spp. contribute approximately half of the
known marine dinitrogen (N
2
) fixation. Rapidly changing environmental factors such as
the rising atmospheric partial pressure of carbon dioxide (pCO
2
) and shallower mixed
layers (higher light intensities) are likely to affect N
2
fixation rates in the future ocean.
Several studies have documented that N
2
fixation in laboratory cultures of T. erythraeum
increased when pCO
2
was doubled from present-day atmospheric concentrations (~380
ppm) to projected future levels (~750 ppm). We examined the interactive effects of light
and pCO
2
on two strains of T. erythraeum Ehrenberg (GBRTRLI101 and IMS101) in
laboratory semi-continuous cultures. Elevated pCO
2
stimulated gross N
2
fixation rates in
cultures growing at 38 µmol quanta·m
-2
·s
-1
(GBRTRLI101 and IMS101) and 100 µmol
quanta·m
-2
·s
-1
(IMS101), but this effect was reduced in both strains growing at 220 µmol
quanta·m
-2
·s
-1
. Effects at higher irradiance were not examined in laboratory cultures of T.
erythraeum but previous fieldwork supports trends reported here. Conversely, CO
2
fixation rates increased significantly (p<0.05) in response to high pCO
2
under mid and
high irradiances only. These data imply that the stimulatory effect of elevated pCO
2
on
CO
2
- and N
2
fixation by T. erythraeum is correlated with light. The ratio of gross:net N
2
fixation was also correlated with light and trichome length in IMS101. Our study
suggests that elevated pCO
2
may have a strong positive effect on Trichodesmium gross
N
2
fixation in intermediate and bottom layers of the euphotic zone, but perhaps not in
light-saturated surface layers. Climate change models must consider the interactive
20
effects of multiple environmental variables on phytoplankton and the biogeochemical
cycles they mediate.
Keywords: carbon dioxide, diazotrophy, light, nitrogen fixation, ocean global change,
Trichodesmium
21
Abbreviations: DIC, dissolved organic carbon; Fe, iron; N, nitrogen; P, phosphorus;
pCO
2
, partial pressure of carbon dioxide; ppm, parts per million
22
Chapter 1 Introduction
Dinitrogen (N
2
) fixation by marine diazotrophic cyanobacteria such as
Trichodesmium spp. contributes substantial new nitrogen (N) to marine environments,
including the North Atlantic, Pacific and Indian oceans (Capone et al. 2005, Karl et al.
2002, Capone et al. 1997, Carpenter et al. 1993). This new nitrogen represents an
important sink for atmospheric carbon dioxide (CO
2
), as organic carbon and nitrogen are
drawn down from surface layers to the deep ocean by the biological carbon pump
(Capone et al. 1997, Karl et al. 1997, Falkowski et al. 1997). For this reason, interest in
diazotrophic production has led to an improved understanding of environmental factors
that control N
2
fixation by marine diazotrophs (Boyd et al. 2010). For instance,
phosphorus (P) and iron (Fe) have been identified as key factors that control N
2
fixation
in the open ocean (Hutchins and Fu 2008). Studies in the North Atlantic Ocean have
demonstrated that P limits N
2
fixation by Trichodesmium spp. in this region (Wu et al.
2000, Sañudo-Wilhelmy et al. 2001, Kustka et al. 2003, Dyhrman et al. 2006, Sohm et al.
2008, Webb et al. 2007). Iron has been suggested as another potential limiting factor for
N
2
fixation by marine diazotrophs (Berman-Frank et al. 2001, Webb et al. 2001, Wu et al.
2000, Fu and Bell 2003, Moore et al. 2009), and in some regimes Fe/P co-limitation may
be important (Mills et al. 2004).
Recently, some studies have suggested that the partial pressure of CO
2
(pCO
2
) in
the atmosphere may be another possible limiting factor for N
2
- and CO
2
fixation by
Trichodesmium (Hutchins et al. 2007, Barcelos e Ramos et al. 2007, Levitan et al. 2007,
2010, Kranz et al. 2009) and the widespread unicellular marine diazotroph, Crocosphaera
23
watsonii (Fu et al. 2008). This finding may be related to the fact that the cyanobacterial
RUBISCO enzyme is relatively inefficient at fixing inorganic carbon (C) compared to
those of many other species of oxygenic photoautotrophs (Badger et al. 1998, Tortell
2000). As in many taxonomic groups of phytoplankton, however, some cyanobacteria
employ carbon-concentrating mechanisms to overcome this limitation (Badger et al.
2006; Price et al. 2008). Currently, atmospheric pCO
2
is approximately 35% higher than
during the pre-industrial era, and continued anthropogenic CO
2
emissions are expected to
double the current concentration (~385 ppm) before the turn of the century (Alley et al.
2007-IPCC). The concomitant increase in pCO
2
in the surface of the world’s oceans
could enhance global N
2
fixation rates, thereby augmenting current inputs of new
nitrogen and potentially increasing net CO
2
draw down (Hutchins et al. 2009). Such
negative feedback mechanisms between N
2
fixation and atmospheric CO
2
have been
hypothesized to be driven by iron supply to oligotrophic regions of the world’s oceans
and are thought to occur on glacial-interglacial time scales (Falkowski 1997, Michaels et
al. 2001). This new evidence suggests that both short term and geological time scale
models of ocean/atmosphere biogeochemical feedbacks may also need to incorporate the
effects of CO
2
limitation of marine N
2
fixation.
Interactive effects of environmental factors that control N
2
fixation are important
to consider because some factors may act synergistically, while others have independent
effects (Boyd et al. 2010). Hutchins et al. (2007) demonstrated that elevated pCO
2
enhanced CO
2
and N
2
fixation rates independently of changes in temperature or P-
limitation of N
2
fixation in Trichodesmium spp. In contrast, the effects of pCO
2
on CO
2
-
24
and N
2
fixation rates in Crocosphaera watsonii (WH8501) were dependent on Fe
availability (Fu et al. 2008). Collectively, these studies indicate that the interactive effects
of multiple environmental factors such as CO
2
, temperature and nutrient concentrations
are important to consider in order to accurately predict how CO
2
- and N
2
fixation rates
will change in diazotrophic cyanobacteria within the next century.
Within the next 50-100 years higher sea surface temperature, increased
precipitation, and ice melting are expected to create a more stratified water column across
much of the world’s oceans. The net effect will decrease the average depth of the mixed
layer, thereby potentially increasing the average irradiance experienced by phytoplankton
(Boyd and Doney 2002, Sarmiento et al. 2004, Behrenfeld et al. 2006, Breitbarth et al.
2007, Boyd et al. 2010). Despite this prediction, the interactive effects of changing light
environments and pCO
2
on CO
2
- and N
2
fixation rates in marine diazotrophs have only
been examined in one other study (results published separately in Kranz et al. 2010 and
Levitan et al. 2010), although several studies have investigated the effects of irradiance
alone. In two different laboratory cultures of T. erythraeum N
2
fixation rates increased
with increasing light and reached a saturation point near 70 µmol quanta·m
-2
·s
-1
(Bell and
Fu 2003) and 100 µmol quanta·m
-2
·s
-1
(Staal et al. 2007). In natural blooms of
Trichodesmium in the tropical North Atlantic, however, N
2
fixation rates trichome
-1
increased with increasing irradiance with maximal rates near the sea surface, where
irradiance may be >1000 µmol quanta·m
-2
·s
-1
(Capone et al. 2005). An inverse
relationship between mixed layer depth and colonial N
2
fixation rates also suggested that
Trichodesmium diazotrophy was limited by light in this region (Sañudo-Wilhelmy et al.
25
2001). Irradiance may partially control the distribution of Trichodesmium in the water
column, such that colonies become negatively buoyant during light hours following
production of carbohydrates and positively buoyant at night following carbohydrate
exudation or consumption (Villareal and Carpenter 2003, Berman-Frank et al. 2007,
Kranz et al. 2009). In a recent study, Davis and McGillicuddy (2006) presented data
suggesting that Trichodesmium may also occupy deep water (>120 meters depth).
This study examines the effects of present-day and elevated pCO
2
(near 100-year
predicted levels) on CO
2
- and N
2
fixation by Trichodesmium erythraeum isolates from
the Pacific (GBRRLI101) and Atlantic (IMS101) oceans, grown under a range of
irradiance. Regardless of geographical origin, both of these Trichodesmium isolates
showed similar responses of CO
2
fixation and gross N
2
fixation rates, suggesting that the
interactive influence of irradiance and elevated pCO
2
needs to be considered to accurately
predict how changing pCO
2
could affect N
2
fixation rates in the future ocean.
26
Chapter 1 Materials and Methods
Cultures- Stock and experimental cultures of Trichodesmium erythraeum
GBRTRL101 (GBR; from the Great Barrier Reef, Pacific Ocean, Fu and Bell 2003) and
IMS101 (IMS; from coastal North Carolina, Atlantic Ocean, Prufert-Bebout et al. 1993)
were cultured at 24ºC (unless otherwise stated) in an artificial seawater medium without
fixed N using a modified version of the YBCII recipe of Chen et al. (1996). Phosphate
and trace metal solutions were filtered (0.2 µm) and added in concentrations equivalent to
the AQUIL recipe (Morel et al. 1979) to micro-wave- (experiment with GBR) or
autoclave-sterilized (experiments with IMS) seawater. The AQUIL concentrations of
phosphate and trace metals are more than sufficient to support maximal growth in our
experiments, where medium is renewed frequently through the semi-continuous dilutions.
Irradiance was supplied with cool white fluorescent bulbs on a 12:12 light:dark cycle.
For all experiments, cultures were grown in triplicate using a semi-continuous batch
culturing method to achieve steady state exponential growth for approximately 7-10
generations prior to sampling, in order to fully acclimatize cells to treatment pCO
2
and
irradiance conditions. We monitored cell density every 2-3 days using microscopic cell
counts. When the biomass reached approximately 100-200 trichomes mL
-1
(~100-200
nmol C·mL
-1
), we diluted cultures with fresh medium to 50-100 trichomes mL
-1
(~50-100
nmol C·mL
-1
). In this semi-continuous culturing method, the growth rate determines the
dilution rate; this culturing technique does not attempt to control the growth rate with the
dilution rate, as continuous culturing methods do.
27
Experimental Design- IMS growth vs. light experiment- This experiment served as
a reference for determining the range of irradiance levels used in our CO
2
and light
experiments. Cultures of IMS were cultured at 27ºC in 15 800-mL culturing flasks on a
12:12 light:dark cycle at 25, 50, 100, 180 and 300 µmol quanta·m
-2
·s
-1
irradiance. Once
cells had achieved steady state, we determined cellular growth rates using microscopic
cell counts between dilutions.
CO
2
/light experiments- Two separate experiments were conducted with two T.
erythraeum strains: GBR and IMS. Cultures of Trichodesmium sp. (GBR) were grown in
12 1-L polycarbonate bottles at 35 and 220 µmol quanta·m
-2
·s
-1
at two concentrations of
CO
2
(see below). In the experiment with IMS, cultures were grown in 18 1-L
polycarbonate bottles at 38, 100 and 220 µmol quanta·m
-2
·s
-1
at two concentrations of
CO
2
. Both experiments were conducted on a 12:12 light:dark cycle at 24ºC.
Within each irradiance treatment for both experiments, cultures were bubbled
with 0.2 µm filtered lab air (for the experiment with IMS) or pre-mixed air (prepared by
Gilmore Liquid Air Company, South El Monte, CA, USA) containing present-day (380
ppm certified value for the experiment with GBR) and elevated, 100-year predicted (750
ppm certified value for both CO
2
experiments) atmospheric CO
2
concentrations. The rate
of bubbling was visually monitored daily to ensure that cultures were bubbled with
sufficient positive gas flow to keep the pH of the cultures at an appropriate level for
respective CO
2
treatments. Based on rates of gas utilization from the supply cylinders,
estimated gas flow rates were between 30-60 mL·min
-1
. Although we did not examine
trichomes for apical cells, we have not encountered detrimental effects of gentle bubbling
28
on trichome length in our previous experiments with culturing Trichodesmium in this
manner (Hutchins et al. 2007, 2009). Because bubbling rates were approximately the
same in all replicates, this factor did not have a differential effect between treatments.
Due to incubator availability constraints in the laboratory, the light manipulation
and the CO
2
/light manipulation experiments were conducted at slightly different
temperatures. While we do not have growth and N
2
fixation rates in response to
temperature for GBR, rates of Trichodesmium isolate IMS are not significantly different
from each other at 24°C and 27°C (Breitbarth et al. 2007).
Analytical Methods- Growth rates – Growth rates were estimated by measuring
relative increases in cell number per unit volume between dilutions (2-3 day periods) in
steady-state cultures.
N
2
fixation – We estimated N
2
fixation rates with two methods: the acetylene
reduction method and the
15
N
2
isotope tracer method. For the acetylene reduction
method, described in Capone (1993), two 10-mL samples from each experimental
replicate were incubated under treatment-specific conditions of irradiance and
temperature in air-tight vials for ~10 hours (starting from the beginning of the light
period) with 2 mL acetylene in 16.75 mL of headspace. The amount of ethylene
accumulation was then estimated in 200 µL headspace gas with a gas chromatograph
(model: GC-8A, Shimadzu Scientific Instruments, Columbia, MD, USA) at the 2
nd
, 4
th
,
6
th
, and 8
th
hour of the light period yielding 3 two-hour rates of ethylene accumulation.
The accumulation of ethylene over 8 hours was used to calculate total gross N
2
fixation
rates (see explanation below) using a conversion ratio of 3:1 for acetylene to N
2
29
reduction. Maximum gross N
2
fixation rates were determined by finding the maximum
rate of ethylene accumulation over a ~2-hour period. To calculate the concentration of
ethylene in seawater from the concentration in the vial headspace, we used the Bunsen
coefficient (0.088; from Breitbarth et al. 2004) at 24°C and a salinity of 35. We also
estimated N
2
fixation rates with the
15
N
2
isotope tracer method (Mulholland and
Bernhardt 2005; Mulholland et al. 2004) by injecting 160 µL of highly enriched (99%)
15
N
2
gas into combusted (4 hours, 450ºC) 159 mL gas tight bottles filled with culture
(without headspace) from a treatment replicate (triplicate samples for each experimental
treatment). Culture samples were then incubated for 12 hours under treatment-specific
conditions of irradiance and temperature during the light period only and we terminated
incubations by filtering samples onto pre-combusted (450ºC, 4 hours) Whatman GF/F
filters (GE Healthcare, Little Chalfont, Buckinghamshire, UK). pH was not determined
in culture subsamples that were used to estimate N
2
fixation after the incubation period in
our experiments. Samples were stored frozen and dried before analysis with a Europa
20/20 isotope ratio mass spectrometer (originally manufactured by: Europa Scientific
Inc., Cincinnati, OH, refurbished by: PDZ Europa Limited, Hill Street, Elworth,
Sandbach, Cheshire, UK) equipped with an automated nitrogen and carbon analyzer
(ANCA). Estimates of N
2
fixation made with the isotope tracer (
15
N
2
) method should
represent net N
2
fixation rates, because in general, it has been shown to estimate fixed N
that is retained within cells (Mulholland et al. 2004). We assumed that the acetylene
reduction method estimates gross N
2
fixation rates because this estimate includes N fixed
regardless of fate (see Mulholland & Bernhardt 2005; Mulholland et al. 2004). We
30
estimated net
15
N
2
fixation rates in our experiment with IMS, but not in our experiment
with GBR.
Carbon fixation - Cell-specific CO
2
fixation rates were determined as in Fu et al.
(2008). Specifically, we inoculated two 30 mL samples from each treatment replicate
with 25 or 50 µL of 1 mCurie (mCi) stock solution of sodium bicarbonate (H
14
CO
3
-
;
0.83-1.7 µCi·mL
-1
final concentration). Samples were incubated for 24 hours under
treatment-specific conditions of irradiance and temperature and then filtered onto
Whatman GF/F filters and rinsed 3 times with ~5 mL filtered seawater to remove
extracellular H
14
CO
3
-
. Non-photosynthetically driven
14
C incorporation was determined
by incubating replicate culture samples (30 mL) for 24 hours in opaque bottles at the
experimental temperature with the same concentration of H
14
CO
3
-
; these values were
subtracted from measured total
14
C incorporation to estimate photosynthetic
incorporation. The total radioactivity of H
14
CO
3
-
was determined by stabilizing 25 or 50
µL of the 1 mCi H
14
CO
3
-
with 100 µL of a basic solution of phenylethylamine (99%)
before adding 4 mL of Ultima Gold
®
XR (PerkinElmer, Shelton CT, USA) Radioactivity
of
14
C was determined on a Multi-purpose Scintillation Counter (model: LS-6500,
Beckman Coulter, Fullerton, CA, USA). Carbon fixation rates (moles of CO
2
fixed per
unit time) were estimated by calculating the ratio of the radioactivity of
photosynthetically-driven
14
C incorporation into cells over 24 hours to the total
radioactivity of H
14
CO
3
-
and multiplying that ratio by the total dissolved inorganic carbon
concentration (DIC, see method below).
31
Seawater carbonate system estimates- Total dissolved inorganic carbon (DIC)
was preserved in whole water samples (5-70 mL; stored at 4°C) with a 5% HgCl
2
solution (final concentration diluted to 0.5% HgCl
2
) as described in Fu et al. (2007), and
estimated by acidifying 5 mL and quantifying the CO
2
trapped in an acid sparging
column (model: CM 5230) with a carbon coulometer (model: CM 140, UIC inc., Joliet,
IL, USA) as described in Beman et al. (2010) and King et al. (2011). Reference material
for the DIC analysis was prepared by Andrew Dickson at Scripps Institute of
Oceanography. pH was measured with a pH meter (model: Orion 5 star Thermo
Scientific, Beverly, MA, USA) and was monitored to ensure that perturbations of the
seawater with the either air or certified pre-mixed air (Gilmore Liquid Air Company 750
ppm) resulted in the desired target pH of either ~8.2 or ~7.95. For the CO
2
/light
manipulation experiment with GBR, samples for total DIC were taken from cultures at
the same time CO
2
and N
2
fixation rates were estimated. For the CO
2
/light manipulation
experiment with IMS, measurements of pH were paired with total DIC samples 5-6 days
prior to measuring rates of CO
2
- and N
2
fixation and were used to calculate pCO
2
at 24°C
using the CO
2
sys program provided by Lewis and Wallace (1998) with K
1
and K
2
constants from Mehrbach et al. (1973) refit by Dickson and Millero (1987).
Cellular C, N and P content – Particulate N and C were estimated in cells filtered
onto combusted GF/F filters. Samples were dried at 80-90°C for 2 days, compressed into
pellets and the amounts of C and N were determined using an elemental analyzer (model:
4010, Costech Analytical Technologies Inc., Valencia, CA, USA). Particulate P was
estimated by filtering 20-30 mL of the cultures onto combusted GF/F filters. Filters were
32
rinsed twice with 2 mL of a 0.17M sodium sulfate solution and dried in a combusted
glass vial with 2 mL of a 0.017M magnesium sulfate solution, as described in Fu et al.
(2005). Samples were then combusted for 2 hours at 450ºC to volatilize organic
compounds bound to P. Residual P was estimated using the spectrophotometric method
of Fu et al. (2005) with a spectrophotometer (model: SP-830, Barnstead/Turner,
Dubuque, Iowa, USA) at a wavelength of 885 nm.
Trichome length –In our light/CO
2
manipulation experiment with IMS, trichome
length was measured with an ocular micrometer in samples collected from treatment-
specific acclimated cultures 2 days prior to and on the final sampling day. Length data
from the two time points were averaged and the resulting mean value from each
experimental replicate was used in our analyses.
Statistics- With JMPIN 4.0.3 statistical software (SAS Institute, Inc. Crary, NC,
USA), we used a one-way and a two-way analysis of variance (ANOVA) test combined
with a Tukey analysis of multiple comparisons to determine statistical differences
(p<0.05) between treatments. We report the standard error (SE) associated with the mean
of treatment replicates.
33
Chapter 1 Results
Seawater carbonate estimates- In the experiment with IMS101, total DIC
concentrations and pH were close to those documented for present-day atmospheric pCO
2
(Table 1). For example, Bates (2001) reported a seasonal range of 2010-2070 µmoles
total CO
2
kg
-1
seawater in the Sargasso Sea for the years 1988-1998. Riebesell et al.
(2010) provides reference values of seawater pH for present-day and 100-year predicted
concentrations of atmospheric CO
2
. Using the certified value of the pre-mixed air and
assuming that the air treatment was 380 ppm, we back-calculated pH with our estimates
of total DIC. Our pH electrode measurements were close to the back-calculated pH
values. We did not measure pH in our experiment with GBR because of instrument
problems and therefore we do not have calculated values of pCO
2
for this experiment;
however, the measured DIC concentrations were close to values expected based on
respective pCO
2
treatments (Table 1).
Growth rates- For both GBR and IMS, growth was limited by light in our low
light treatments (irradiance of 25-35 µmol quanta·m
-2
·s
-1
; growth rates of 0.13-0.16 d
-1
)
compared to light-saturated maximum growth rates of 0.25-0.45 d
-1
at irradiance ≥100
µmol quanta·m
-2
·s
-1
(Fig. 1a-c). Within pCO
2
treatments for IMS, growth rates at 220
µmol quanta·m
-2
·s
-1
were not significantly different than those at 100 µmol quanta·m
-2
·s
-1
(p>0.15). The whole model effect of the two-way ANOVA indicated that elevated pCO
2
had a significant positive effect on cellular growth in IMS (p=0.05, F
1,17
=4.6; Fig. 1b) but
not in GBR (p=0.37, F
1,11
=0.88; Fig. 1c).
34
Carbon fixation- Irradiance and pCO
2
had a significant impact on C-specific CO
2
fixation rates in both strains of T. erythraeum (p<0.02) (Figs. 2a, 3a). Elevated pCO
2
enhanced C-specific CO
2
fixation rates at irradiances ≥100 µmol quanta·m
-2
·s
-1
in both
IMS (p<0.001, F
1,12
>40) and GBR (p=0.01, F
1,8
=10.7) but not under low light (IMS
p=0.92, F
1,12
<0.01; GBR p>0.36, F
1,8
=0.94). Cell-normalized CO
2
fixation rates yielded
similar results, with significantly different rates between pCO
2
treatments at irradiances
≥100 µmol quanta·m
-2
·s
-1
in both strains (p<0.03) but not at low light in either strain
(p>0.09) (Figs.2b, 3b). Thus, elevated pCO
2
had no effect on CO
2
fixation at low light
but had a positive effect at high light.
N
2
fixation- The effects of light and pCO
2
on gross N
2
fixation rates in IMS and
GBR were examined in four ways; maximum and total gross N
2
fixation rates (ethylene
production rates; see Methods) were normalized to both cellular N and cell density. Net
N
2
-fixation rates (
15
N
2
uptake rates) were also normalized to cellular N and cell density.
Not surprisingly, light had a highly significant impact on gross N
2
fixation rates in
IMS and GBR, and on net N
2
fixation rates in IMS (p<0.001) (Figs. 2c-h, 3c-f). Total and
maximum gross N
2
fixation rates at 220 µmol quanta·m
-2
·s
-1
were not significantly higher
than rates measured in treatments maintained at 100 µmol quanta·m
-2
·s
-1
in either pCO
2
treatment (p>0.10, F
1,12
<3.2, Figs 2c-f). Conversely, net N
2
fixation rates were
significantly higher in IMS cultures maintained at 220 µmol quanta·m
-2
·s
-1
than at 100
µmol quanta m
-2
s
-1
in both pCO
2
treatments (p<0.01, F
1,12
>9.6, Fig. 2 g,h). Thus, while
steady-state gross N
2
fixation rates were saturated near 100 µmol quanta·m
-2
·s
-1
at both
35
pCO
2
levels investigated, net N
2
fixation rates may not have been saturated, even at 220
µmol quanta·m
-2
·s
-1
.
Elevated pCO
2
had a highly significant effect on total N-specific gross N
2
fixation
rates in IMS, according to the whole two-way ANOVA model (p<0.001, F
1,12
=62, Fig.
2c), but in GBR, increases were smaller, variability between replicates was higher, and
differences were not significant (p>0.71, F
1,8
=0.15, Fig. 3c). Cell-density-normalized
total gross N
2
fixation rates were stimulated by high pCO
2
in IMS only at irradiance ≥100
µmol quanta·m
-2
·s
-1
(p<0.01, F
1,12
>12.8, Fig. 2d), and not in the low or high light
treatments with GBR (p=0.12, F
1,8
=3.0, Fig. 3d). Likewise, elevated pCO
2
significantly
increased maximum N-specific gross N
2
fixation rates in IMS at irradiance ≥100 µmol
quanta·m
-2
·s
-1
(p<0.003, F
1,12
>14, Fig. 2e). Maximum cell-specific gross N
2
fixation
rates in IMS, however, were significantly higher in the elevated pCO
2
treatment
compared with the present-day pCO
2
treatment at all irradiances examined (p<0.01,
F
1,12
>9.6, Fig. 2f). Elevated pCO
2
did not significantly affect maximum gross N
2
fixation
rates in GBR when N
2
fixation was normalized to cellular N (p>0.06, F
1,8
<4.6, Fig. 3e),
but had a significant positive effect at low light when normalized to cell numbers
(p=0.48, F
1,8
=5.4, Fig. 3f). There were no significant differences in N-specific net
15
N
2
fixation rates between pCO
2
treatments in IMS (p=0.68, F
1,12
=0.18; Fig. 2g), but pCO
2
had a significant positive effect on net N
2
fixation rates (
15
N
2
-uptake) when normalized to
cell density at 220 µmol quanta·m
-2
·s
-1
(p=0.01, F
1,11
=9.9, Fig. 2h).
Cellular C, N, and P content - IMS cellular C content varied as a function of light
(p=0.01, F
2,11
=7.3) but not as a function of pCO
2
(p=0.7, F
1,11
=0.16, Fig. 4a). Average
36
cellular N concentrations were higher in the high pCO
2
treatments compared with
present-day pCO
2
treatments at irradiance ≥100 µmol quanta·m
-2
·s
-1
, although not
significantly (p>0.05; Fig. 4b). Cellular P content was significantly higher in the high
pCO
2
treatment relative to the present-day pCO
2
treatment (p>0.03, Fig. 4c) but did not
vary as a function of light (p>0.05).
Gross:Net N
2
fixation- The ratio of total gross:net N
2
fixation gives an estimate of
cellular N release (Mulholland et al. 2004, Mulholland 2007). For IMS, this ratio was
significantly higher in the high pCO
2
treatment compared to the low pCO
2
treatment
under low irradiance (p<0.001, F
1,12
=38), but not at irradiance ≥100 µmol quanta·m
-2
·s
-1
(p>1.8, F
1,12
<2.0) (Fig. 5a). Total gross:net N
2
-fixation was negatively correlated with
light (r=-0.85).
Trichome length – The partial pressure of CO
2
had a strong effect on trichome
length in IMS (p=0.007, F
1,12
=10). The low-light (p=0.003, F
1,12
=14) and mid-light (0.02,
F
1,12
=6.6) treatments contributed to the pCO
2
treatment variation, with trichomes being
longer under the present-day pCO
2
treatment compared with high pCO
2
(Fig. 5b); pCO
2
did not affect trichome length at high-light (p=0.5, F
1,12
=0.5). In addition, the effect of
elevated pCO
2
on the percent increase in trichome length was negatively correlated with
the effect of elevated pCO
2
on the percent increase in total gross:net N
2
-fixation rates
(r=0.72, n=3, Fig. 5), and highly correlated with light (r=0.98).
37
Chapter 1 Discussion
The main finding of our study is that the effect of elevated pCO
2
on gross
and
net
N
2
fixation rates from Trichodesmium erythraeum was dependent on light intensity. The
results from the CO
2
/light experiments with two different strains of T. erythraeum were
similar. In both strains of T. erythraeum, our data indicate that the positive effect of
elevated pCO
2
on gross N
2
fixation was large (~50% increase) under mid and/or low
irradiances compared with that at high light (~20% increase; Figs. 2 and 6). Data from
Kranz et al. (2010), Levitan et al. (2010) and our unpublished field experiments (see
below) corroborate our laboratory data describing the combined effects of light and
elevated pCO
2
on N
2
fixation rates by IMS and GBR. We speculate that the declining
effect of elevated pCO
2
on N
2
fixation with increasing light could have been a negative
feedback interaction caused by enhanced retention of cellular N under high light
conditions (see description below).
We determined the effect of elevated pCO
2
on maximum and total gross N
2
fixation rates in IMS and GBR by calculating the percent increase in rates in high pCO
2
treatments over rates in present-day pCO
2
treatments (Fig. 6). To factor in both cellular
nitrogen and cell density as separate estimators of biomass, we averaged the effect of
elevated pCO
2
on gross N
2
fixation (% change) when gross N
2
fixation was normalized to
cell number and the effect when gross N
2
fixation was normalized to cellular N. We
supplemented our GBR data set on the effect of elevated pCO
2
on gross N
2
fixation rates
with data from two experiments reported by Hutchins et al. (2007). In those experiments,
the maximum chlorophyll a-normalized gross N
2
fixation rates were 54% and 63% higher
38
in elevated pCO
2
treatments relative to current-day pCO
2
treatments in cultures growing
under conditions similar to those used in our experiments (24ºC, 100 µmol quanta·m
-2
·s
-
1
; Fig. 6). These data indicate that the effect of elevated pCO
2
on maximum and total
gross N
2
fixation rates was negatively correlated with light in IMS (r=-0.98, -0.68
respectively). In GBR the effect of elevated pCO
2
on maximum gross N
2
fixation rates
was also negatively correlated with light (r= -0.85). We also calculated the effect of
elevated pCO
2
on net
15
N
2
fixation rates in the same way in IMS. When normalized to
both cell number and cellular N the effect was positively correlated with irradiance,
increasing with increasing light intensity (r=0.85 and 0.84 respectively; Fig. 6).
Three field experiments also indicated that net N
2
fixation rates (
15
N
2
incorporation) increased in response to elevated pCO
2
at high irradiance in a deck-board
incubation of natural Trichodesmium colonies collected from the Gulf of Mexico
(Hutchins et al. 2009). However, gross N
2
fixation rates (acetylene reduction) in these
experiments did not increase with pCO
2
(unpublished data). In those shipboard
experiments, colonies were collected from near the surface and the incubation irradiance
was high. Both the net and gross estimates of N
2
fixation rates follow our conceptual
model in Fig. 6 if the trends were extended to near surface irradiance. However, our lab
experimental data extend only as high as 220 µmol quanta·m
-2
·s
-1
, so the general trends
in these parameters at very high surface light intensities (>1000 µmol quanta·m
-2
·s
-1
)
remain speculative.
Kranz et al. (2010) reported similar combined effects of irradiance and elevated
pCO
2
on gross N
2
fixation rates in IMS 101. Under low light (50 µmol quanta·m
-2
·s
-1
)
39
gross N
2
fixation rates were 200% higher in a high pCO
2
treatment (900 µatm) compared
with a low pCO
2
treatment (150 µatm), whereas under high light (200 µmol quanta·m
-2
·s
-
1
) gross N
2
fixation rates were only 112% higher under elevated pCO
2
compared with a
low pCO
2
treatment. Collectively, all of these studies indicate that the effect of elevated
pCO
2
on gross N
2
fixation rates in Trichodesmium is high under low irradiance and that
this effect decreases as light increases.
At elevated pCO
2
, reduced demand for active transport of inorganic carbon across
the cell membrane may shift additional energy to N
2
fixation in Trichodesmium. Several
authors have hypothesized that elevated pCO
2
may indirectly enhance N
2
fixation rates in
diazotrophic cyanobacteria by this mechanism (Barcelos e Ramos et al. 2007, Hutchins et
al. 2007, Levitan et al. 2007, Fu et al. 2008, Kranz et al. 2009). Badger et al. (2006) and
Price et al. (2008) report that Trichodesmium actively transports HCO
3
-
across the cell
membrane with low to medium affinity transporters, and lacks high affinity carbon
transporters. Kranz et al. (2009), however, indicated that K
1/2
values for HCO
3
-
changed
in response to pCO
2
, perhaps due to differential expression or post-translational
modification of medium and low affinity HCO
3
-
transporters. Our experiments appear to
support the energy shift hypothesis at mid and low irradiances, but our laboratory and
field data suggest that this effect on gross N
2
fixation rates diminishes at high light.
One possible explanation for the declining effect of elevated pCO
2
on gross N
2
fixation rates as irradiance increased is that oxygen production was higher in the high
light, high pCO
2
treatment relative to the high light, present-day pCO
2
treatment. The
reduced effect of elevated pCO
2
on N
2
fixation rates at high light might then be attributed
40
to the well-documented inhibitory effect of oxygen on the nitrogenase enzyme (Gallon
1981, Zehr et al. 1993). We discount this explanation, however, as Kranz et al. (2010)
reported convincing evidence that O
2
production did not increase with pCO
2
in their high
light treatments with IMS 101.
Another possible cause for this declining effect of elevated pCO
2
on gross N
2
fixation rates at high light is differences in intracellular N concentration between pCO
2
treatments. This hypothesis assumes that intracellular N concentration exerts an
influence on gross N
2
fixation rates through a negative feedback response. Indeed, the
cellular N content in our experiment with IMS increased with increasing light (Fig. 4b),
with the highest cellular N concentration in the high light, high pCO
2
treatment. We
speculate that this higher intracellular N concentration may have caused the reduced
gross N
2
fixation rates in this treatment.
At high light, our cellular N content data corroborate findings by Kranz et al.
(2010) showing that PON production increased as a function of pCO
2
. We speculate that
the relatively high accumulation of N in the high pCO
2
treatment probably had an
inhibitory effect on N
2
fixation, and data from Levitan et al. (2010) and Kranz et al.
(2010) seem to support this hypothesis. In their experiment, low concentrations of the
nitrogenase Fe protein (NifH) were associated with high PON production rates in the
high light, high pCO
2
treatment, whereas high NifH concentrations were associated with
low PON production rates in a low pCO
2
treatment. Those data suggest that high PON
levels at high irradiance may influence a negative feedback response on NifH at the
transcription level. At low irradiance, our gross N
2
fixation data are similar to the high
41
effect of elevated pCO
2
on gross N
2
fixation reported by Kranz et al. (2010). As
discussed above, the reason for the high gross:net ratio in our high pCO
2
treatment at low
light with IMS is unknown. The lack of a pronounced effect of elevated pCO
2
on net
15
N
2
uptake rates we documented at low light (Fig. 2g) does not seem to be consistent
with the increased PON production rates at elevated pCO
2
at low irradiance reported by
Kranz et al. (2010).
Interestingly, both trichome length and irradiance seemed to be related to
cellular N retention in IMS (Fig. 5). For instance, when trichome length did not change
as a function of light, as in the high pCO
2
treatment, the gross:net ratio declined with
increasing irradiance, implying that cellular N retention increased with increasing light
(see Mulholland et al. 2004, Mulholland et al. 2007). In the present-day pCO
2
treatment,
trichome length varied dramatically with light, increasing with decreasing irradiance. In
this case, the gross:net ratio remained low at all light levels, implying that cellular N
retention did not change as a function of light. Although we do not know why trichome
length was so dramatically different among pCO
2
treatments at low irradiance in our
experiment with IMS, these data suggest that trichome length and cellular N retention
might be related in some way. Conversely, Levitan et al. (2007) reported an increase in
average trichome length with increasing pCO
2
in IMS, although differences were not
significant between pCO
2
treatments, and that experiment was conducted at 80 µmol
quanta·m
-2
·s
-1
. The largest impact of elevated pCO
2
on trichome length in our
experiment with IMS was at a considerably lower irradiance of 38 µmol quanta·m
-2
·s
-1
.
Although the gross:net N
2
fixation rate ratio is correlated with trichome length data in our
42
CO
2
/light experiment with IMS, the physiological reason for this relationship is unclear.
Unfortunately we do not have estimates of gross:net N
2
fixation rates for GBR and we
did not measure trichome length in that experiment. Future experiments are needed to
investigate this relationship further, particularly at low irradiance.
Elevated pCO
2
did not enhance net cellular CO
2
fixation rates at low light but did
at higher light levels in our light/CO
2
experiment with IMS (Fig. 2a-b). Similarly,
elevated pCO
2
acted to increase cellular growth rates of IMS only under mid and high
irradiance (Fig 1b); growth rates were not different between treatments of pCO
2
under
low light. In contrast, Kranz et al. (2010) reported that the highest effect of elevated pCO
2
on PON production and growth rates was at low irradiance, which was somewhat
proportional to the effect of elevated pCO
2
on gross N
2
fixation rates. In contrast, for the
same IMS strain we found the largest growth rate enhancement by pCO
2
at high light
(Fig. 1b). Our growth rate data for GBR, however, corroborate the combined effects of
light and elevated pCO
2
on growth rates in IMS reported by Kranz et al. (2010) (Fig. 1c).
Growth rates of GBR were higher in the elevated pCO
2
treatment at low light, but were
not different between pCO
2
treatments at high light.
Differences in cellular N retention in response to light and pCO
2
might explain
differences in the response of gross N
2
fixation rates to elevated pCO
2
.between our
experiments with IMS and GBR. For example, elevated pCO
2
had a significant positive
effect on gross N
2
fixation rates by IMS at all light levels when normalized to cellular N
(Fig. 2 c, e). This is not the case for GBR, however; elevated pCO
2
only had a significant
positive effect on maximum gross N
2
fixation rates under low light when normalized to
43
cell density (Fig. 3f). Differences in cellular N retention could have influenced the
cellular N content between strains, and may be responsible for the differing effects of
elevated pCO
2
on gross N
2
fixation rates.
We assume that the ratio of acetylene reduction to N
2
reduction did not vary
substantially among our treatments of light and pCO
2
. Various studies have used different
ratios for Trichodesmium and other cyanobacteria (Carpenter et al. 1977, Mague et al.
1977, Montoya et al. 1996). For consistency we used the acetylene:nitrogen reduction
ratio of 3:1, which has been used in our previous studies involving Trichodesmium IMS
and GBR (Hutchins et al. 2007). While the precision of this ratio might be important for
determining N retention and exudation when examining the gross:net N
2
fixation rates in
field studies, we were mostly interested in making comparisons between culture
experimental treatments. The assumption that the ratio of acetylene:nitrogen reduction
does not change between treatments of pCO
2
and light may or may not be valid, and this
question needs to be examined in future studies.
Even though our estimates of gross and net N
2
fixation rates were calculated over
different amounts of time (10 hours for gross and 12 hours for net estimates), incubation
times for both estimates were centered on the middle of the light period, where N
2
fixation rates are highest (Berman Frank et al. 2001, Kranz et al. 2009). For example, our
gross N
2
fixation rates probably accounted for more than 90% of the total N
2
fixed. Still,
our gross rates are probably slightly higher than they would have been if they were
documented over a 12-hour period, thus affecting our calculated gross:net N
2
fixation rate
44
ratios. Minor differences in this ratio would however probably not affect our
interpretation of the data.
Steady-state cell-specific growth rates in our experiments with IMS were
saturated near 100 µmol quanta·m
-2
·s
-1
(Fig. 1 panels a and b), while Goebel et al. (2009)
and Breitbarth et al. (2008) report an IMS growth saturation point near 140 µmol
quanta·m
-2
·s
-1
and 180 µmol quanta·m
-2
·s
-1
, respectively. One important distinction that
separates our study from those other two studies is that growth kinetic data in our study
were collected from steady-state, semi-continuous batch cultures, whereas those studies
used batch cultures in non-steady state growth. Although we do not have enough growth
rate data in response to light for GBR to predict a light saturation point for growth, our
data suggest that the maximum steady-state growth rate for GBR is near 0.45 d
-1
(Fig. 1).
Similar to results reported by Staal et al. (2007), gross N
2
fixation rates were saturated
near 100 µmol quanta·m
-2
·s
-1
in our CO
2
/light experiment with IMS in both low and high
pCO
2
cultures. In addition, the impact of elevated pCO
2
on gross N
2
fixation rates in
response to elevated pCO
2
was greatest at 100 µmol quanta·m
-2
·s
-1
.
The colonial form often dominates natural Trichodesmium blooms rather than free
trichomes. Our laboratory cultures did not contain colonies, but instead consisted of free
trichomes. The physiological responses of colonial Trichodesmium to elevated pCO
2
and
changing irradiance might be different than the responses of free trichomes, and further
studies with intact natural colonies are needed.
Future field studies should examine the influence of elevated pCO
2
on CO
2
and
N
2
fixation at higher light intensities. Our results suggest that elevated pCO
2
may not
45
have a positive effect on gross N
2
fixation rates at surface irradiance, which would yield a
lower net effect of rising pCO
2
on global N
2
fixation rates than previously estimated.
Instead, the effect of elevated pCO
2
on gross N
2
fixation rates may be more important at
lower light intensities as suggested in our conceptual model in Figure 6. Capone et al.
(1994) suggested that a major pathway of N and C transfer from Trichodesmium through
trophic levels might be by amino acid leakage from Trichodesmium cells, followed by
assimilation by heterotrophs. If elevated pCO
2
and light can influence cellular N retention
in the open ocean, this finding may have consequences for the ecology and
biogeochemistry of future oceans. For instance, models predict higher average irradiance
experienced by phytoplankton circulating within shallower mixed layers and modest
increases in primary productivity (Breitbarth et al. 2007, Boyd and Doney 2002,
Sarmiento et al. 2004, Boyd et al. 2010). Finally, it is evident that future studies
investigating the impacts of elevated pCO
2
on CO
2
- and N
2
fixation in Trichodesmium
should include field experiments that encompass the entire mixed layer.
46
Chapter 1 Acknowledgements
We thank Eric Webb for allowing us to use his gas chromatograph for the acetylene
reduction assay, and Doug Capone for allowing us to use some of his general lab
equipment. Grant support was provided by NSF OCE 0722337 to D. Hutchins, NSF
OCE 0850730 to F.-X. Fu, and NSF OCE 0722395 to M. Mulholland.
47
Chapter 1 References
Alley, R.B., Berntsen, T., Bindoff, N.L., Chen, Z. and others. Summary for
policymakers. In: Solomon S, Qin D, Manning M, Chen Z and others (Eds.) Climate
change 2007: The physical science basis. Contribution of Working Group I to the fourth
assessment report of the Intergovernmental Panel on Climate Change. Cambridge
University Press, Cambridge and New York. (2007).
Badger, M. R., Andrews, T. J., Whitney, S. M., Ludwig, M., Yellowlees, D. C., Leggat,
W. & Price, G. D. 1998. The diversity and coevolution of RubisCO, plastids, pyrenoids,
and chloroplast based CO
2
-concentrating mechanisms in algae. Can. J. Bot. 76:1052-
1071.
Badger, M. R., Price, G. D., Long, B. M. & Woodger, F. J. 2006. The environmental
plasticity and ecological genomics of the cyanobacterial CO
2
concentrating mechanism.
J. Exp. Bot. 57:249–265.
Barcelos e Ramos, J., Biswas, H., Schulz, K. G., LaRoche, J. & Riebesell, U. 2007.
Effect of rising atmospheric carbon dioxide on the marine nitrogen fixer Trichodesmium.
Glob. Biogeochem. Cycles 21:GB2028.
Bates, N. R. 2001. Interannual variability of oceanic CO
2
and biogeochemical properties
in the Western North Atlantic subtropical gyre. Deep-Sea Res. II 48:1507-1528.
Bell, P. R. F. & Fu, F-X. 2003. Effect of light on growth, pigmentation and N
2
fixation of
cultured Trichodesmium sp. from the Great Barrier Reef lagoon. Hydrobiologia 543:25–
35.
Behrenfeld, M. J., O’Malley, R. T., Siegel, D. A., McClain, C. R., Sarmiento, J. L.,
Feldman, G. C., Milligan, A. J., Falkowski, P. G., Letelier, R. M., & Boss, E. S. 2006.
Climate-driven trends in contemporary ocean productivity. Nature 444:752-755.
Beman, J. M., Chow, C.-E., King, A. L., Feng, Y., Fuhrman, J. A., Anderson, A. Bates,
N. R., Popp, B. N. & Hutchins, D. A. 2010. Global declines in oceanic nitrification rates
as a consequence of ocean acidification. PNAS, USA. 108:208-213.
Berman-Frank, I., Lundgren, P., Chen, Y-B., Kupper, H., Kolber, Z., Bergman, B. &
Falkowski, P. 2001. Segregation of nitrogen fixation and oxygenic photosynthesis in the
marine cyanobacterium Trichodesmium. Science 294:1534-1537.
Berman-Frank, I., Quigg, A., Finkel, Z. V., Irwin, A. J. & Haramaty, L. 2007. Nitrogen-
fixation strategies and Fe requirements in cyanobacteria. Limnol. Oceanogr. 52:2260–
2269.
48
Boyd, P. W. & Doney, S. C. 2002. Modeling regional responses by marine pelagic
ecosystems to global climate change. Geophys. Res. Lett. 29:1806.
Boyd, P. W., Strzepek, R., Fu, F.-X. & Hutchins, D.A. 2010. Environmental control of
open ocean phytoplankton groups: now and in the future. Limnol. Oceanogr.
55: 1353–
1376.
Breitbarth, E., Mills, M. M., Friedrichs, G. & LaRoche, J. 2004. The Bunsen gas
solubility coefficient of ethylene as a function of temperature and salinity and its
importance for nitrogen fixation assays. Limnol. Oceanogr.: Methods 2:282-288.
Breitbarth, E., Oschlies, A., & LaRoche J. 2007. Physiological constraints on the global
distribution of Trichodesmium - effect of temperature on diazotrophy. Biogeosciences.
4:53-61.
Breitbarth, E., Wohlers, J., Klas, J., LaRoche, J. & Peeken, I. 2008. Nitrogen fixation
and growth rates of Trichodesmium IMS-101 as a function of light intensity. Mar. Ecol.
Prog. Ser. 359:25-36.
Capone, D. G. 1993. Determination of nitrogenase activity in aquatic samples using the
acetylene reduction procedure. In: Kemp, P. F., J. J. Cole, B. F. Sherr, E. B. Sherr (eds).
Handbook of methods in aquatic microbial ecology. Lewis Publishers, Boca Raton, FL, p
621–631.
Capone, D. G., Ferrier, M. D., & Carpenter, E. J. 1994. Amino acid cycling in colonies of
the planktonic marine cyanobacterium Trichodesmium thiebautii. Appl. Env. Microbiol.
60:3989-3995.
Capone, D. G., Zehr, J. P., Paerl, H. W., Bergman, B. & Carpenter, E. J. 1997.
Trichodesmium, a globally significant marine cyanobacterium. Science 276:1221-1229.
Capone, D. G., Burns, J. A., Montoya, J. P., Subramaniam, A., Mahaffey, C., Gunderson,
T., Michaels, A. F. & Carpenter, E. J. 2005. Nitrogen fixation by Trichodesmium spp.:
An important source of nitrogen to the tropical and subtropical North Atlantic Ocean.
Global Biogeochem. Cy. 19:GB2024.
Carpenter, E. J. & C. C. Price. 1977. Nitrogen fixation , distribution and production of
Oscillatoria (Trichodesmium) spp. in the western Sargasso and Caribbean Seas. Limnol.
Oceanogr. 22:60-72.
Carpenter, E. J., O’Neil, J. M., Dawson, R., Capone, D. G., Siddiqui, P. J. A.,
Roenneberg, G. T. & Bergman, B. 1993. The tropical diazotrophic phytoplanktonkter
Trichodesmium: biological characteristics of two common species. Mar. Ecol. Prog. Ser.
95:295-304.
49
Chen, Y. B., Zehr, J. P. & Mellon, M. 1996. Growth and nitrogen fixation of the
diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp IMS 101
in defined media: Evidence for a circadian rhythm. J. Phycol. 32:916-923.
Davis, C. S. & McGillicuddy, D. J. 2006. Transatlantic abundance of the N
2
-fixing
colonial cyanobacterium Trichodesmium. Science 312:1517-1519.
Dickson, A. G., & Millero, F. J. 1987. A comparison of the equilibrium constants for the
dissociation of carbonic acid in seawater media. Deep-Sea Res. 34:1733-1743.
Dyhrman S. T., Chappell, P. D., Haley, S. T., Moffett, J. W., Orchard, E. D., Waterbury,
J. B. & Webb, E. A. 2006. Phosphonate utilization by the globally important marine
diazotroph Trichodesmium. Nature 439:1452-1458.
Falkowski, P. G. 1997. Evolution of the nitrogen cycle and its influence on the biological
sequestration of CO
2
in the ocean. Nature 387:272-275.
Fu, F.-X. & Bell, P. R. F. 2003. Growth N
2
-fixation and photosynthesis in a
cyanobacterium, Trichodesmium sp., under Fe stress. Biotech. Lett. 25, 645-649.
Fu, F.-X., Zhang, Y., Bell, P. R. F. & Hutchins, D. A. 2005. Phosphate uptake and
growth kinetics of Trichodesmium (Cyanobacteria) isolates from the North Atlantic
Ocean and the Great Barrier Reef, Australia. J. Phycol. 41:62-73.
Fu, F-X., Warner, M. E., Zhang, Y., Feng, Y. & Hutchins, D. A. 2007. Effects of
increased temperature and CO
2
on photosynthesis, growth and elemental ratios in marine
Synechococcus and Prochlorococcus (Cyanobacteria). J. Phycol. 43:485-496.
Fu, F.-X., Mulholland, M. R., Garcia, N. S., Beck, A., Bernhardt, P. W., Warner, M. E.,
Sanudo-Wilhelmy, S. A. & Hutchins, D. A. 2008. Interactions between changing pCO
2
,
N
2
fixation, and Fe limitation in the marine unicellular cyanobacterium Crocosphaera.
Limnol. Oceanogr. 53:2472-2484.
Gallon, J. R. 1981. The oxygen sensitivity of nitrogenase: a problem for biochemists and
micro-organisms. Trends Biochem. Sci., 6, 19-23.
Goebel, N. L., Edwards, C. A., Carter, B. J., Achilles, K. M., & Zehr, J. P. 2008. Growth
and carbon content of three different-sized diazotrophic cyanobacteria observed in the
subtropical North Pacific. J. Phycol. 44:1212-1220.
Hutchins, D. A., Fu, F. X., Zhang, Y., Warner, M. E., Feng, Y., Portune, K., Bernhardt,
P. W. & Mulholland, M. R. 2007. CO
2
control of Trichodesmium N
2
-fixation,
50
photosynthesis, growth rates, and elemental ratios: Implications for past, present, and
future ocean biogeochemistry. Limnol. Oceanogr. 52:1293-1304.
Hutchins, D. A. & Fu, F.-X. 2008. Linking the oceanic biogeochemistry of iron and
phosphorus with the marine nitrogen cycle. pp. 1627-1653. In: Nitrogen in the Marine
Environment, 2
nd
edition. D. G. Capone, D. A. Bronk, M. R. Mulholland and E. J.
Carpenter [Eds.], Elsevier Press, Amsterdam.
Hutchins, D. A., Mulholland, M. R., & Fu, F.-X. 2009. Nutrient cycles and marine
microbes in a CO
2
enriched ocean. Oceanography 22:128-145.
Karl, D., Letelier, R., Tupas, L., Dore, J., Christian, J. & Hebel, D. 1997. The role of
nitrogen fixation in biogeochemical cycling in the subtropical North Pacific Ocean.
Nature 388:533-538.
Karl, D., Michaels, A., Bergman, B., Capone, D., Carpenter, E., Letelier, R., Lipschultz,
F., Paerl, H., Sigman, D. & Stal, L. 2002. Dinitrogen fixation in the world’s oceans.
Biogeochem. 57/58:47-98.
King, A. L., Sanudo-Wilhelmy, S. A., Leblanc, K., Hutchins, D. A. & Fu, F.-X. 2011.
CO
2
and vitamin B
12
interactions determine bioactive trace metal requirements of a
subarctic Pacific diatom. ISME 5:1388-1396. doi:10.1038/ismej.2010.211.
Kranz, S. A., Sultemeyer, D., Richter, K. U., & Rost, B. 2009. Carbon acquisition by
Trichodesmium: The effect of pCO
2
and diurnal changes. Limnol. Oceanogr. 54:548-559.
Kranz, S. A., Levitan, O., Richter, K-U., Prášil, O., Berman-Frank, I., Rost, B. 2010.
Combined effects of CO
2
and light on the N
2
-fixing cyanobacterium Trichodesmium
IMS101: Physiological responses. Plant Physiol. 154:334-345.
Kustka, A. B., Sanudo-Wilhelmy, S. A., Carpenter, E. J., Capone, D., Burns, J. & Sunda,
W. G. 2003. Iron requirements for dinitrogen- and ammonium-supported growth in
cultures of Trichodesmium (IMS 101): Comparison with nitrogen fixation rates and iron:
carbon ratios of field populations. Limnol. Oceanogr. 48:1869-1884.
Levitan, O., Rosenberg, G., Šetlík, I., Setlikova, E., Grigel, J., Klepetar, J., Prasil, O. &
Berman-Frank, I. 2007. Elevated CO
2
enhances nitrogen fixation and growth in the
marine cyanobacterium Trichodesmium. Glob. Change Biol. 13:531-538.
Levitan, O., Kranz, S. A., Spungin, D., Prášil, O., Rost, B. & Berman-Frank, I. 2010.
Combined effects of CO
2
and light on the N
2
-fixing cyanobacterium Trichodesmium
IMS101: A mechanistic view. Plant Physiol. 154:346-356.
51
Lewis, E. and D. W. R. Wallace (1998). Program Developed for CO
2
System
Calculations. ORNL/CDIAC-105. Carbon Dioxide Information Analysis Center, Oak
Ridge National Laboratory, U.S. Department of Energy, Oak Ridge, Tenessee. Available
at: http://cdiac.ornl.gov/oceans/co2rprt.html (last accessed 20 March 2011).
Mague, T. H., Mague, C., & Holm-Hansen, O. 1977. Physiology and chemical
composition of nitrogen-fixing phytoplankton in the central North Pacific Ocean. Mar.
Biol. 24:109-119.
Mehrbach, Y., Culberson, C., Hawley, J. & Pytkovicz, R. 1973. Measurement of the
apparent dissociation constants of carbonic acid in seawater at atmospheric pressure.
Limnol. Oceanogr. 18:897-907.
Michaels, A. F., Karl, D. M. & Capone, D. G. 2001. Element stoichiometry, new
production and nitrogen fixation. Oceanography 14:68–77.
Mills, M. M., Ridame, C., Davey, M., La Roche, J. and Geider, R. J. 2004. Iron and
phosphorus co-limit nitrogen fixation in the eastern tropical North Atlantic. Nature
429:292-294.
Montoya, J. P., Voss, M., Kähler, P. & Capone, D. G. 1996. A simple, high-precision,
high-sensitivity tracer assay for N
2
fixation. Appl. and Environ. Microbiol. 62:986-993.
Moore, C. M., Mills, M.M., Achterberg, E.P.,Geider, R.J., LaRoche, J., Lucas, M.I.,
McDonagh, E.L., Pan, X., Poulton, A.J., Rijkenberg, M. J. A., Suggett, D.J., Ussher, S.J.,
Woodward, E.M.S. 2009. Large-scale distribution of Atlantic nitrogen fixation
controlled by iron availability. Nature Geoscience 12: 867-871.
Morel, F. M. M., Rueter, J. G., Anderson, D. M. and Guillard, R. R. L. 1979. Aquil -
chemically defined phytoplankton culture-medium for trace-metal studies. J. Phycol.
15:135-141.
Mulholland, M. R. 2007. The fate of nitrogen fixed by diazotrophs in the ocean.
Biogeosciences Discuss. 4:37-51.
Mulholland, M. R. and Bernhardt, P. W. 2005. The effect of growth rate, phosphorus
concentration and temperature on N
2
-fixation, carbon fixation, and nitrogen release in
continuous cultures of Trichodesmium IMS101. Limnol. Oceangr. 50:839-849.
Mulholland, M. R., Bronk, D. A. & Capone, D. G. 2004. N
2
fixation and regeneration of
NH
4
+
and dissolved organic N by Trichodesmium IMS101. Aquatic Microb. Ecol. 37:85-
94.
52
Price, G. D., Badger, M. R., Woodger, F. J. & Long B. M. 2008. Advances in
understanding the cyanobacterial CO
2
-concentrating-mechanism (CCM): functional
components, Ci transporters, diversity, genetic regulation and prospects for engineering
into plants. J. Exp. Bot. 59:1441–1461.
Prufert-Bebout, L., Paerl, H. W., & Lassen, C. 1993. Growth, nitrogen fixation, and
spectral attenuation in cultivated Trichodesmium species. Appl. Env. Microb. 59:1367-
1375.
Riebesell, U., Fabry, V. J., Hansson, L., Gattuso, J. P. 2010. Guide to best practices for
ocean acidification research and data reporting. Publications Office of the European
Union, Luxembourg, 258 pp.
Sanudo-Wilhelmy, S. A., Kustka, A. B., Gobler, C. J., Hutchins, D. A., Yang, M., Lwiza,
K., Burns, J., Capone, D. G., Raven, J. A. & Carpenter, E. J. 2001. Phosphorus limitation
of nitrogen fixation by Trichodesmium in the central Atlantic Ocean. Nature 411: 66-69.
Sarmiento, J. L., Slater, R., Barber, R., Bopp, L., Doney, S. C., Hirst, A. C., Kleypas, J.,
Matear, R., Mikolajewicz, U., Monfray, P., Soldatov, V., Spall, S. A. & Stouffer, R.
2004. Response of ocean ecosystems to climate warming. Global Biogeochem. Cy.
18:GB3003. doi:10.1029/2003GB002134.
Sohm, J. A., Mahaffey, C. & Capone, D. G. 2008. Assessment of relative phosphorus
limitation of Trichodesmium spp. in the North Pacific, North Atlantic, and the north coast
of Australia. Limnol. Oceanogr. 53:2495-2502.
Staal, M., Rabouille, S. & Stal, L. J. 2007. On the role of oxygen for nitrogen fixation in
the marine cyanobacterium Trichodesmium sp. Environ. Microbiol. 9:727-736.
Stramma, L., Johnson, G. C., Sprintal, J. & Mohrholz, V. 2008. Expanding oxygen-
minimum zones in the tropical oceans. Science 32:655-658.
Tortell, P. D. 2000. Evolutionary and ecological perspectives on carbon acquisition in
phytoplankton. Limnol. Oceanogr. 45:744-750.
Villareal, T. A. & Carpenter, E. J. 2003. Buoyancy regulation and the potential for
vertical migration in the oceanic cyanobacterium Trichodesmium. Microb. Ecol. 45:1–10.
Webb, E. A., Moffett, J. W. & Waterbury J. B. 2001. Iron stress in open-ocean
cyanobacteria (Synechococcus, Trichodesmium, and Crocosphaera spp.): Identification
of the IdiA protein. Appl. Environ. Microbiol. 67:5444–5452.
53
Webb, E. A., Jakuba, R. W., Moffett, J. W. & Dyhrman, S. T. 2007. Molecular
assessment of phosphorus and iron physiology in Trichodesmium populations from the
western Central and western South Atlantic. Limnol. Oceanogr. 52:2221–2232.
Wu, J. F., Sunda, W., Boyle, E. A. & Karl, D. M. 2000. Phosphate depletion in the
western North Atlantic Ocean. Science 289:759-762.
Zehr, J. P., Wyman, M., Miller, V., Duguay, L. & Capone, D. G. 1993. Modification of
the Fe protein of nitrogenase in natural populations of Trichodesmium thiebautii. Appl.
Environ. Microbiol. 59:669-676.
54
Chapter 1 Table 1.
55
Chapter 1 Figures
Chapter 1 Figure 1. Cellular growth rate of Trichodesmium erythraeum (IMS101 and
GBRRLI101) in response to irradiance (panel a) and present-day and elevated pCO
2
(panels b-c). Growth rates were estimated from changes in cell number per unit volume
over time. The standard error is reported on the means of triplicate samples.
Chapter 1 Figure 1.
56
Chapter 1 Figure 2. CO
2
- and N
2
fixation rates by Trichodesmium erythraeum (IMS101)
at present-day (air) and elevated pCO
2
(750 ppm) under variable irradiance. C-specific
CO
2
fixation (panel a) and cell-specific CO
2
fixation (panel b). Total N-specific gross N
2
fixation (panel c), total cell-specific gross N
2
fixation (panel d), maximum N-specific
gross N
2
fixation (panel e), maximum cell-specific gross N
2
fixation (panel f), net N-
specific N
2
fixation rates (panel g), and net cell-specific N
2
fixation rates (panel h). C-
and N-specific rates are in units of hr
-1
, and cell-specific rates are in units of fmol C or
N·cell
-1
·hr
-1
. The standard error is reported on the means of triplicate samples.
Chapter 1 Figure 2.
57
Chapter 1 Figure 3. Response of CO
2
and N
2
fixation in Trichodesmium erythraeum
(GBRRLI101) to present-day and elevated pCO
2
under variable irradiance. CO
2
fixation
was normalized to cellular C (panel a, hr
-1
) and cell density (panel b, fmol C·cell
-1
·hr
-1
).
Total (8-hour rate, panels c-d) and maximum (highest 2-hour rate, panels e-f) gross N
2
fixation rates were normalized to cellular N (hr
-1
) and cell density (fmol N·cell
-1
·hr
-1
).
The standard error is reported on the means of triplicate samples.
Chapter 1 Figure 3.
58
Chapter 1 Figure 4. Cellular quotas of carbon (panel a), nitrogen (panel b) and
phosphorus (panel c), in present-day (closed symbols) and elevated pCO
2
(open symbols)
cultures of Trichodesmium erythraeum IMS101. The standard error is reported on the
means of triplicate samples.
Chapter 1 Figure 4.
59
Chapter 1 Figure 5. Total (8-hour rate) gross:net (12-hour rate) N
2
-fixation (panel a) and
trichome length (µm, panel b) in present-day and elevated pCO
2
cultures of
Trichodesmium erythraeum IMS101 as a function of irradiance. The standard error is
reported on the means of triplicate samples.
Chapter 1 Figure 5.
60
Chapter 1 Figure 6. Postulated effect of elevated pCO
2
on N
2
fixation in Trichodesmium
erythraeum IMS101 and GBRRLI101 in a water column setting expressed as a percent
increase from present-day pCO
2
. Estimates of the effect were normalized to cell density
and cellular N and averaged for the maximum (highest 2-hour rate) and total (8-hour rate)
gross N
2
fixation rates and the net
15
N
2
fixation (12-hour) rates. *Effect of elevated pCO
2
(present-day to 750 ppm pCO
2
) on chlorophyll-a-normalized maximum N
2
fixation rate
of GBR from Hutchins et al. (2007) at 100 µmol quanta·m
-2
·s
-1
. The standard error is
reported on means. Light data were paired with depth data from Breitbarth et al. (2008).
Question marks estimate published (net
15
N
2
fixation) and unpublished (gross N
2
fixation) results from field experiments with Trichodesmium colonies collected near
surface waters (see Hutchins et al. 2009).
Chapter 1 Figure 6.
61
Chapter 2
COMBINED EFFECTS OF CO
2
AND IRRADIANCE ON LARGE AND SMALL
ISOLATES OF THE UNICELLULAR N
2
-FIXING CYANOBACTERIUM
CROCOSPHAERA WATSONII FROM THE WESTERN TROPICAL ATLANTIC
OCEAN
Manuscript prepared for European Journal of Phycology
Nathan S. Garcia
Department of Biological Sciences, University of Southern California, Los Angeles, CA
90089, USA
Fei-Xue Fu
Department of Biological Sciences, University of Southern California, Los Angeles, CA
90089, USA
Cynthia L. Breene
Department of Biological Sciences, University of Southern California, Los Angeles, CA
90089, USA
Elizabeth K. Yu
Department of Biological Sciences, University of Southern California, Los Angeles, CA
90089, USA
Peter W. Bernhardt
Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University,
Norfolk, VA 23529, USA
Margaret R. Mulholland
62
Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University,
Norfolk, VA 23529, USA
David A. Hutchins
2
Department of Biological Sciences, University of Southern California, Los Angeles, CA
90089, USA
2
Corresponding author; dahutch@usc.edu
63
Chapter 2 Abstract
We examined the combined effects of irradiance and pCO
2
on growth, N
2
- and CO
2
-
fixation rates in a strain with small (WH0401) and large (WH0402) cells of the
unicellular marine N
2
-fixing cyanobacterium Crocosphaera watsonii that were isolated
from the western tropical Atlantic Ocean. In low-pCO
2
-acclimated cultures (190 ppm) of
WH0401, growth, CO
2
- and N
2
-fixation rates were significantly lower than those
acclimated to higher (present-day ~385 ppm, or future ~750 ppm) pCO
2
treatments
(p≤0.02). These rates, however, were not significantly different in low-pCO
2
-acclimated
cultures of WH0402 in comparison with higher pCO
2
treatments. Unlike previous reports
for C. watsonii (WH8501), N
2
-fixation rates did not increase further in cultures of either
isolate when acclimated to 750 ppm relative to those maintained at present-day pCO
2
.
Both irradiance (p<0.001) and pCO
2
(p<0.04) had a significant negative effect on
gross:net N
2
-fixation rates in WH0402 and trends were similar in WH0401, implying that
retention of fixed N was enhanced under elevated irradiance and pCO
2
. These data,
along with previously reported results, suggest that C. watsonii may have wide-ranging,
strain-specific responses to changing irradiance and pCO
2
, emphasizing the need for
examining the effects of global change on a range of isolates within this
biogeochemically-important genus. In general, however, our data suggest that cellular N
retention and CO
2
-fixation rates of C. watsonii may be positively affected by elevated
light and pCO
2
within the next 100 years, potentially increasing trophic transfer
efficiency of C and N and thereby facilitating uptake of atmospheric carbon by the
marine biota.
64
Keywords: carbon dioxide, Crocosphaera, cyanobacteria, diazotroph, light, nitrogen
fixation, ocean global change, unicellular
65
Chapter 2 Introduction
Within the past two decades, emerging data have suggested that the magnitude of
marine N
2
fixation has been grossly underestimated (Deutsch et al. 2007; Capone 2008;
Mulholland et al. in press). Traditionally, Trichodesmium has been widely accepted as a
major contributor to oceanic N
2
fixation, however, estimates of N
2
fixation by unicellular
diazotrophs continue to increase and their calculated N inputs to marine systems may
narrow the gap in the global N budget (Zehr et al. 2001; Montoya et al. 2004; Church et
al. 2008; Moisander et al. 2010). Understanding how these key components of the
marine N cycle will respond to rapid global change is essential to predict how the carbon
cycle will change.
In the next 100 years, anthropogenic inputs of CO
2
to the atmosphere will likely
double the present-day partial pressure of CO
2
(pCO
2
). At the same time, the average
global mixed layer depth is also expected to decrease, thereby contributing to higher
mean irradiances experienced by phytoplankton (Sarmiento et al. 2004; Behrenfeld et al.
2006; Boyd et al. 2010). In addition to their individual effects, we now recognize that
interactive effects of these and other environmental factors must be considered to
realistically predict the net impacts of global change (Hutchins et al. 2007; Hutchins et al.
2009; Fu et al. 2008; Kranz et al. 2010; Levitan et al. 2010; Garcia et al. 2011).
Crocosphaera watsonii is a model organism that has been widely studied and represents
oceanic unicellular photosynthetic N
2
fixers (Zehr et al. 2001; Zehr et al. 2007). In this
study, we examined how two isolates of this genus might respond to global change.
66
Previous studies suggest that elevated pCO
2
acts to enhance gross N
2
-fixation
rates by the oceanic diazotrophs Trichodesmium erythraeum and C. watsonii (Barcelos e
Ramos et al. 2007; Hutchins et al. 2007; Levitan et al. 2007; Fu et al. 2008). Recently,
however, several experiments indicated that light moderated the effect of elevated pCO
2
on gross N
2
fixation by T. erythraeum (Kranz et al. 2010; Garcia et al. 2011). Elevated
pCO
2
acted to enhance N
2
-fixation rates under low irradiances but this stimulatory effect
was reduced at high light, which may have been caused by an enhanced ability to retain
newly fixed cellular N at high irradiances (Garcia et al. 2011).
To date, the majority of published work investigating physiological responses of
C. watsonii has focused on one strain (WH8501) and physiological studies of other
strains are currently lacking. Recently, Webb et al. (2009) compared N
2
-fixation rates by
strains of C. watsonii with small cells vs. strains with large cells. In that study, chl a-
normalized N
2
-fixation rates in a large strain isolated from the North Pacific Ocean
(WH0003) were twice as high as those in a smaller strain isolated from the South Atlantic
Ocean (WH8501). In this regard, some of our preliminary data suggested that the effects
of elevated pCO
2
on N
2
fixation by C. watsonii were different between strains and that
this difference may be dependent on cell size among strains. Thus, a major goal of this
study is to examine the effects of CO
2
on growth, CO
2
- and N
2
-fixation rates of two
isolates of C. watsonii from the western tropical Atlantic Ocean in laboratory culture
experiments under a range of irradiances: one with small cells (2-3 µm diameter;
WH0401) and an isolate with large cells (4-6 µm diameter; WH0402). Our specific aim is
67
to collect data that might aid in predicting how N and C cycles and trophic structure will
be impacted as a result of climate change.
68
Chapter 2 Methods
Culturing and experimental design - Stock cultures of the two Atlantic C.
watsonii isolates used in this study were provided courtesy of Dr. Eric Webb. Both
isolates were collected in March 2002; WH0401 from 6º 58.78 N, 49º 19.70 W and
WH0402 from 11º42.12S, 32º00.64W. An outline of all experiments with both isolates,
including experimental variables used for each, is presented in Table 1. In all
experiments, triplicate cultures were grown using a semi-continuous culturing technique
(Garcia et al. 2011) at 28°C in an artificial seawater medium (Chen et al. 1996). Nutrients
were added to autoclaved seawater at the concentrations listed in the AQUIL recipe
(Morel et al. 1979), except for nitrate, which was omitted. The growth rates of cultures
were measured over 2-3 day intervals and were used to determine the dilution rate.
Culture cell density was kept low (cells ml
-1
= 50-500 x 10
3
for experiments with
WH0401 and 5.0-30 x 10
3
for WH0402; Table 1) to prevent light limitation of
photosynthesis and deviation from the expected pH values for respective pCO
2
culture
treatments. Light was supplied with cool white fluorescent lamps on a 12:12 hr
light:dark cycle. Because of large differences in cell size between WH0401 and
WH0402, we cultured WH0401 at higher cell densities (cells mL
-1
) to maintain relatively
equivalent levels of total culture biomass (as cellular carbon). For CO
2
experiments,
media and cultures were bubbled with air (present-day pCO
2
concentration, ~385 ppm) or
premixed air prepared by Gilmore Liquid Air Company with certified values of 190 ppm
pCO
2
(last glacial maximum levels; Petit et al. 1999) and 750 or 761 ppm pCO
2
(within
the range predicted for the year 2100; Alley et al. 2007) for the entire term of the
69
experiment (Table 1). Cells were considered fully acclimated to treatment conditions
after cultures had remained at steady-state growth for more than 7 generations (unless
stated otherwise). Cultures were sampled 24-48 hrs after the last dilution to measure
growth rates, gross and net
15
N
2
-fixation rates, CO
2
-fixation rates, particulate elemental
composition, and carbonate system measurements (for CO
2
experiments).
Light experiments – In order to quantify differences in growth, CO
2
- and N
2
-
fixation rate capacities of these two isolates of C. watsonii, we measured growth, gross
and net N
2
fixation and CO
2
-fixation rates, and particulate elemental composition in
response to a range of light intensities (labeled experiments 1 and 2 in Table 1).
Preliminary CO
2
experiments - We conducted preliminary experiments with C.
watsonii at 3 levels of CO
2
(190, air, and 750 ppm) and an irradiance of 155 µE m
-2
s
-1
(labeled experiments 3 and 4 in Table 1).
CO
2
–light experiments - To determine if light moderates the effect of elevated
pCO
2
on growth, CO
2
- and N
2
-fixation rates of C. watsonii, we first grew WH0402 under
two concentrations of CO
2
(air and 750 ppm) at 5 irradiances (18-300 µmol quanta m
-2
s
-
1
; labeled experiment 6 in Table 1). In this experiment we measured similar growth and
N
2
-fixation rates between these CO
2
concentrations. Therefore, when examining
responses of WH0401 with this experimental design, we added a low CO
2
treatment (190
ppm) under the same range of irradiances (labeled experiment 5 in Table 1). Despite
several attempts, we were not able to acclimate WH0401 to any of these CO
2
concentrations at 18 or 50 µmol quanta m
-2
s
-1
for unknown reasons, but possibly due to a
compromised cell membrane (see Discussion).
70
Analytical Methods- Growth rate and cell density estimates – Growth rates were
determined by enumerating cells microscopically using a hemocytometer. Cell diameter
was measured microscopically using an ocular micrometer. Growth rates were fitted to a
Monod linear hyperbolic function of irradiance (Monod 1949) using Sigma Plot 10
software program. The hyperbola was fitted to the data without including the origin to
yield the highest r
2
value. This best fit method yields more realistic Monod parameters
with a critical threshold of irradiance for growth.
N
2
fixation - For all experiments we used the acetylene reduction assay described
by Capone (1993) as an estimate of the gross N
2
-fixation rate. All rate measurements in
the light and CO
2
-light experiments were initiated at the beginning of the 12-hr dark
period when C. watsonii is known to fix N
2
(Mohr et al. 2010a; Saito et al. 2011). For the
preliminary CO
2
experiments this assay was initiated during the 7
th
hr of the 12-hr dark
period and continued for 4 hrs. For this assay, two 50 ml (light and CO
2
–light
experiments) or 60 ml (preliminary CO
2
experiments) culture samples were collected
from each replicate and incubated in 80 ml cubic polycarbonate bottles at 28°C. Four ml
of acetylene were injected into the headspace ~1 h after the beginning of the dark period
and samples were withdrawn from the headspace every 2-3 hours to measure acetylene
reduction. In the CO
2
–light experiment with WH0401, we measured rates throughout the
dark period and continued to measure them during the early portion of the light cycle,
when samples were exposed to treatment light levels (Table 1). In this experiment, we
gently agitated incubation bottles to equilibrate ethylene in the seawater with ethylene in
the headspace. In other prior experiments, we were not yet aware that this agitation
71
yielded higher rates by forcing this gas equilibrium. Gross N
2
-fixation rates were
calculated in the same way as described in Garcia et al. (2011) using a Bunsen coefficient
for ethylene of 0.082 (Breitbarth et al. 2004) and an ethylene production:N
2
-fixation ratio
of 3:1.
We also measured net N
2
-fixation rates using the
15
N
2
isotope tracer method
(Mulholland & Bernhardt 2005; Mulholland et al. 2004). Samples were prepared the
same way as described in Garcia et al. (2011) and incubated at 28°C in complete darkness
for 12 h during the dark period. At the time we conducted these experiments, we were not
aware of criticisms of the
15
N
2
uptake method that have been discussed by Mohr et al.
(2010b) who provide evidence that
15
N
2
diffusion rates are slow and limit
15
N
2
uptake
rates. Thus, for another independent estimate of net N
2
fixation, we calculated a
particulate N accumulation rate in cultures over time (ΔPN = PN
final
-PN
initial
) by using our
estimates of particulate N. Particulate N was measured in subsamples of experimental
replicates that were incubated with
15
N
2
at the end of the dark period and used as the end-
period PN measurement (PN
final
). Because only one sample of PN was collected, we
back-calculated an estimate of PN
initial
based on our measurements of cellular growth rate
using the equation: growth rate (d
-1
) = [ln(PN
final
)-ln(PN
initial
)]/(t
2
-t
1
), where t
1
is the initial
time and t
2
is the final time. Based on our measurements of growth rates, we assumed that
PN cell
-1
was in a daily steady state. Thus, we used these two different net N
2
fixation
estimates to calculate the gross N
2
-fixation rate:PN-accumulation rate ratio (gross:PN)
and compared it with the gross N
2
-fixation rate:net
15
N
2
-fixation rate ratio (gross:net),
which is a proxy for cellular N retention (Mulholland et al. 2004; Mulholland 2007).
72
CO
2
fixation - The rate of CO
2
fixation was determined as described in Garcia et
al. (2011) using the H
14
CO
3
-
incorporation method. Briefly, 30 mL of each experimental
culture replicate was incubated at 28°C and treatment levels irradiance with 25 or 50 µL
of 1 mCurie (mCi) stock solution of sodium bicarbonate (H
14
CO
3
-
; 0.83-1.7 µCi·mL
-1
final concentration). CO
2
-fixation rates were determined by first calculating the ratio of
radioactivity of cellular incorporation of
14
C during 24 h to the total radioactivity of
H
14
CO
3
-
. This ratio was then multiplied by the total CO
2
concentration (TCO
2
). TCO
2
concentrations were measured in our CO
2
-light experiments (experiments 5 and 6 in
Table 1) and were applied to all experiments to calculate CO
2
-fixation rates for
corresponding CO
2
treatments. For the light experiments (experiments 1 and 2 in Table
1), we used a TCO
2
value that was measured in the present-day pCO
2
treatments of the
CO
2
-light experiments (2053 µM TCO
2
). Samples for the analysis of TCO
2
were
preserved and measured with a carbon coulometer as described in Garcia et al. (2011).
TCO
2
analyses were not available in our preliminary CO
2
experiments (experiments 3
and 4 in Table 1).
Particulate C and N – Culture samples from each experimental replicate (100 ml)
were filtered onto precombusted (450°C, 4 h) GF/F filters for the analysis of cellular N
and C. Filters were then dried at 80-90°C, compressed into pellets and the amounts of C
and N were determined using an elemental analyzer (Costech Instruments, model 4010).
Statistics- The analysis of variance statistical test (two-way and one-way
ANOVA) combined with a Tukey analysis of multiple comparisons was used to
73
determine statistical differences (p<0.05) between treatments. We report the standard
error (SE) associated with the mean of treatment replicates.
74
Chapter 2 Results
Light experiments (experiments 1 and 2) – Mean specific growth rates of
WH0402 were higher than those of WH0401 at all light levels investigated (p<0.05; Fig
1a). Cells of WH0401 were considerably smaller than cells of WH0402 and average cell
diameters were ~20% larger in high-light-acclimated cells compared to low-light-
acclimated cells in both strains (p<0.05) (Fig. 1b). The Monod fit of growth as a function
of light yielded a theoretical maximum growth rate of 0.95 d
-1
(r
2
=0.99) for WH0402 and
0.68 d
-1
(r
2
=0.99) for WH0401. However, the half-saturation constant (K
1/2
) for growth
with respect to light and the critical threshold (T
cr
) for growth were similar between
strains (WH0401, K
1/2
=61 µmol quanta m
-2
s
-1
, T
cr
=11 µmol quanta m
-2
s
-1
; WH0402,
K
1/2
=59 µmol quanta m
-2
s
-1
, T
cr
=13 µmol quanta m
-2
s
-1
). Because of the large
differences in cell size between strains, we compared C-specific CO
2
- and N-specific N
2
-
fixation rates. Both C-specific CO
2
-fixation rates (Fig. 1c) and N-specific gross N
2
-
fixation rates (Fig. 1d) were consistently higher in the strain with large cells (WH0402)
than in the strain with small cells (WH0401), except at the lowest light level, similar to
the pattern of their specific growth rates. Growth rates were highly correlated with N-
specific
15
N
2
-fixation rates (r=0.85, n=5 for WH0401; r=0.99, n=5 for WH0402) (Fig. 1a,
1e). The gross:net N
2
-fixation rate ratios declined with increasing light intensity and
were negatively correlated with specific growth rates (WH0401, r=-0.91, n=4; WH0402,
r=-0.92, n=5) and cell volumes (WH0401, r=-0.89, n=4; WH0402, r=-0.71, n=5; Fig. 1f).
Preliminary CO
2
experiments (experiments 3 and 4)– Results from these
experiments imply that WH0401 and WH0402 have different CO
2
requirements (e.g.
75
K
1/2
) to achieve maximum growth and N
2
fixation rates. Results from the preliminary
experiment with WH0401 should be interpreted cautiously, however, because cell growth
was very slow in all cultures in the initial stage of the experiment and as a result cultures
in the low-pCO
2
treatment (190 ppm) only doubled 1-2 times under the experimental
conditions. This slow growth might represent lag phase growth rather than an acclimated
growth condition. Cells in the air and 750 ppm CO
2
treatments had approximately 7
doublings before sampling. Conversely, growth of WH0402 in the preliminary
experiment was rapid and cells doubled more than 10 times before measuring growth,
CO
2
- and N
2
-fixation rates. Measured pH values in cultures were comparable to bubbled
cultures in our other CO
2
experiments (Table 2). The partial pressure of CO
2
did not have
a significant effect on growth, CO
2
-fixation, N
2
-fixation or PN-accumulation rates of the
isolate with large cells (WH0402; F
2,6
≤ 4.5, p≥0.06; Figs. 2 and 3). Contrastingly, pCO
2
had a strong positive effect on growth, CO
2
-fixation and N
2
-fixation and PN-
accumulation rates by the strain with small cells (WH0401; F
2,6
≥20, p≤0.002; Figs. 2 and
3). Despite this strong effect of low pCO
2
(190 ppm), growth, biovolume-normalized
CO
2
-fixation, gross and net N
2
-fixation and PN-accumulation rates by WH0401 were not
significantly different between the present-day and elevated pCO
2
treatments (F
1,6
=5.1,
p≥0.065, Figs. 2 and 3). Mean gross:net N
2
-fixation rate ratios decreased with increasing
pCO
2
(WH0401: F
2,6
=4.2, p=0.07; WH0402: F
2,6
≥5.8, p≤0.04) and were negatively
correlated with specific growth rates in both isolates (for both isolates r =-0.99, n=3; Fig.
2), suggesting that pCO
2
had a positive effect on cellular N retention. The gross:PN
accumulation ratio was considerably lower than the gross:net N
2
-fixation rate ratio and
76
was not significantly different between pCO
2
treatments in either isolate (F
2,6
≤ 0.7,
p≥0.5; Figs. 2 and 3).
CO
2
-light experiments (experiments 5 and 6) – Measured TCO
2
concentrations
and pH values in our cultures were within the expected range for the respective pCO
2
treatments (Table 2). Specific growth rates of WH0401 were not significantly different
between the two high CO
2
treatments (F
1,18
=0.20, p= 0.66), but growth rates in the two
high CO
2
treatments were significantly higher than those in the 190 ppm pCO
2
treatment
(F
1,18
>55, p<0.001; Fig. 4a). Although not significant, elevated pCO
2
had a slightly
positive effect on mean specific growth rates of WH0402 at all irradiances investigated
except for the 50 µmol quanta m
-2
s
-1
treatment (F
1,20
=3.2, p<0.09; Fig. 4b), most likely
because cultures did not grow well in these bottles for unknown reasons. Cell-
normalized CO
2
-fixation rates were positively affected by pCO
2
in WH0401 (F
2,18
=4.7,
p=0.02), but the interactive effect between light and pCO
2
was not significant (F
4,18
=0.13,
p=0.97; Fig. 4c). Light and pCO
2
, however, did have a significant positive interactive
effect on cellular CO
2
-fixation rates in cultures of C. watsonii WH0402 (F
1,20
=13,
p=0.002; Fig. 4d) indicating that the effect of elevated pCO
2
significantly increased with
increasing light.
Gross cellular N
2
-fixation rates of WH0401 were not affected by light between
100-300 µE m
-2
s
-1
treatments (F
2,18
=3.0, p=0.1), or by pCO
2
between the present-day
and elevated pCO
2
treatments (F
1,18
=0.22, p=0.65), but were significantly reduced in the
190 ppm treatment compared to higher pCO
2
treatments (F
1,18
≥7.8, p≤0.01; Fig. 5a).
Similarly, for WH0402, gross cellular N
2
-fixation rates were not significantly different
77
between the present-day and elevated pCO
2
treatments (F
1,20
=3.1, p=0.09; Fig. 5b), but
significantly increased as a function of increasing light between all light treatments
(F
1,20
>7.2; p<0.02).
Trends in cell-normalized net
15
N
2
-fixation rates by WH0401 were similar to
those observed for growth rates; low pCO
2
had a significant negative effect on net
15
N
2
-
fixation rates in comparison with higher pCO
2
levels (F
1,18
≥8.2, p≤0.01) and rates were
not different between the air and elevated pCO
2
concentrations (F
1,18
=0.2, p=0.67; Fig.
5c). In WH0402, cell-normalized net
15
N
2
-fixation rates were not significantly different
between the air and elevated pCO
2
treatments (F
1,20
=0.08, p=0.77; Fig. 5d), but were
strongly affected by light (F
4,20
=64; p<0.0001). PN-accumulation rates by WH0401 were
lower than gross N
2
-fixation rates but considerably higher than net
15
N
2
-fixation rates
(Fig. 5e). In WH0402, PN-accumulation rates were similar to gross N
2
-fixation rates and
higher than net
15
N
2
-fixation rates (Fig. 5f; see Methods section for methodological
differences in the acetylene assay between experiments). Both light and pCO
2
had
significant positive effects on gross N-specific N
2
-fixation rates by WH0401 (F
2,18
= 26,
p<0.001 F
2,18
=8.0 p=0.003) and differences in gross N-specific N
2
fixation between the
190 ppm pCO
2
treatment and higher pCO
2
treatments were more pronounced in
comparison to gross cell-normalized N
2
-fixation rates (Fig. 5g). In WH0402, gross N-
specific N
2
-fixation rates were not significantly different between pCO
2
treatments and
were light saturated near 100 µmol quanta m
-2
s
-1
(p>0.05, Fig. 5h). In both strains,
trends in N-specific net
15
N
2
-fixation rates (Fig. 5i, j) were very similar to trends in
growth rates (Fig. 4a, b).
78
Both light (F
2,18
, p<0.0001) and pCO
2
(F
2,18
=5.4, p=0.01) had a significant
negative effect on gross:net N
2
fixation in WH0401 but the interactive effect of light and
pCO
2
on the gross:net ratio was not significant (p>0.05; Fig. 6a). In WH0402, light and
pCO
2
did have a significant interactive effect on gross:net N
2
fixation; the effect of
elevated pCO
2
on gross:net N
2
fixation significantly increased with decreasing irradiance
(F
4,20
=3.9, p=0.02; Fig. 6b), suggesting that the effect of elevated pCO
2
on cellular N
retention was strongest under low light. Growth rates of WH0402 were strongly anti-
correlated with the gross:net N
2
-fixation rate ratio (r =-0.95). Light was the most
important controlling factor on the gross:PN accumulation ratio in WH0401 (F
2,18
=3.5,
p=0.05; Fig. 6c); the gross:PN accumulation ratio declined with increasing irradiance. We
interpret this ratio as a separate estimate of the gross:net N
2
fixation ratio and is thus a
proxy for cellular retention of fixed N. While pCO
2
had no effect on the gross:PN
accumulation ratio in WH0402 (F
1,20
=0.17, p=0.69), the two-way ANOVA test suggested
that light had a significant negative effect on this ratio (F
4,20
=5.6, p=0.003; Fig. 6d),
although this was driven mostly by the large increase in the ratio at 50 µmol quanta m
-2
s
-
1
. In both strains, the range of the gross:PN accumulation ratio was substantially lower
than the range of the gross:net N
2
-fixation rate ratio (Fig. 6).
Light period N
2
-fixation rates –Light and pCO
2
had significant positive effects on
gross N
2
-fixation rates during the early part of the light period in our CO
2
-light
experiment with WH0401 (experiment 5). In all three pCO
2
treatments, gross N
2
-fixation
rates during this time were higher in high-light treatments (180 and 300 µE m
-2
s
-1
) when
compared with the 100 µE m
-2
s
-1
treatment (p<0.05; Fig. 7). In all three irradiance
79
treatments, gross N
2
-fixation rates were higher in the present-day and elevated pCO
2
treatments in comparison with the 190 ppm pCO
2
treatment (p<0.05) during this time.
When compared with the dark period, gross N
2
-fixation rates declined during the early
light period in all pCO
2
treatment cultures maintained at 100 µE m
-2
s
-1
and in the 190
ppm pCO
2
treatments at all irradiances (Fig. 7). In the light-saturated treatments (180 and
300 µE m
-2
s
-1
), ~20-25% of all fixed nitrogen was fixed during the light period in the
present-day and 761 ppm pCO
2
treatments, compared with only ~10-15% in the 190 ppm
pCO
2
treatment.
80
Chapter 2 Discussion
Our results identified both similarities and differences in physiological responses
to changing pCO
2
and irradiance between large and small strains of Crocosphaera
watsonii isolated from the western equatorial region of the Atlantic Ocean. In our light
experiments, maximum growth responses were different between strains but other Monod
functional growth responses (K
1/2
and T
cr
) were similar between strains. Overall, our data
indicate that the strain with larger cells (WH0402) had higher growth, N
2
-, and CO
2
-
fixation rates at near-saturating irradiance when compared to the strain with smaller cells
(WH0401), despite having similar photosynthetic efficiencies at high light (data not
shown). These high rates for WH0402 may give this strain an ecological advantage in
regions of the ocean where nutrient concentrations are relatively high, whereas the
smaller WH0401 strain, with a higher surface area:cell volume ratio, may be better able
to survive in lower-nutrient oceanic waters. In both strains, however, cell diameter was
highly plastic under a range of light intensities, with high-light acclimated cells being
~20% larger than those acclimated to low light.
Our findings do not support previous data regarding CO
2
effects on specific
growth rates of C. watsonii (South Atlantic strain WH8501 from 28°S, 48°W; Fu et al.
2008); in both strains, mean specific growth rates were not significantly different
between the present-day and elevated pCO
2
treatments under any of the light levels that
we tested in our experiments. Growth rates of WH0401, however, were significantly
reduced under low pCO
2
(190 ppm) at all light levels (100-300 µmol quanta m
-2
s
-1
),
whereas growth rates of WH0402 were only slightly reduced at low pCO
2
near saturating
81
irradiance (155 µmol quanta m
-2
s
-1
). These data suggest that WH0402 has a low K
1/2
for
growth with respect to pCO
2
in comparison to WH0401, and also that the present-day
concentration of pCO
2
is near growth-saturating levels for both of these strains.
Collectively, however, these data support the notion that there may be significant
differences among strains of Crocosphaera in terms of their growth requirements for
CO
2
.
Elevated pCO
2
leads to high N
2
-fixation rates in Trichodesmium (Hutchins et al.
2007; Barcelos e Ramos et al. 2007; Levitan et al. 2007, Kranz et al. 2009). This effect
may be explained by higher rates of diffusion of CO
2
across the cell membrane under
high pCO
2
conditions, thereby decreasing the energy demand associated with the active
transport of bicarbonate (HCO
3
-
) (Barcelos e Ramos et al. 2007; Hutchins et al. 2007;
Levitan et al. 2007), the main source of inorganic carbon used to supply CO
2
fixation in
Trichodesmium and Crocosphaera (Badger et al. 2006; Price et al. 2008). This “extra”
energy can then be used to support high CO
2
-fixation rates in the early portion of the light
period, leading to larger photosynthate reserves under elevated pCO
2
. This large carbon
reserve can then be respired in the middle of the light period and the resulting energy can
be used to fix N
2
when both respiration and N
2
-fixation rates are maximal and CO
2
-
fixation rates are low (Berman Frank et al. 2001; Kranz et al. 2009). Therefore, N
2
fixation possibly benefits from elevated pCO
2
by two mechanisms: an increase in energy
derived from respiration of a large photosynthate reserve and an increase in energy
derived from the light reactions, indirectly caused by the reduced energy demand
associated with HCO
3
-
transport. The benefit to N
2
fixation associated with reduced
82
HCO
3
-
transport is possible because CO
2
-fixation rates are always positive during the
light period (Kranz et al. 2009).
This model for the effect of elevated pCO
2
on N
2
fixation in Trichodesmium may
be similar for Crocosphaera with respect to larger photosynthate reserves and
consequential respiration of those reserves under high pCO
2
. For instance, Saito et al.
(2011) documented an increase in cellular glycogen content during the light period in C.
watsonii (WH8501), which was likely respired during the following dark period when N
2
fixation was maximal. Because N
2
fixation is primarily a dark process in Crocosphaera,
however, the majority of N
2
fixation cannot receive the more direct energetic benefit
associated with HCO
3
-
transport, as in Trichodesmium, because CO
2
fixation and HCO
3
-
transport are driven by light (Badger et al. 2006; Price et al. 2008). Thus, the indirect
effect of elevated pCO
2
on N
2
fixation by Crocosphaera seems to be to allow the cell to
accumulate larger glycogen reserves during the preceding light period and the energy
acquired from respiration of those reserves can then be used to drive N
2
fixation during
the dark hours. Hence, because only one mechanism is assumed to be involved during the
majority of the N
2
fixation period for Crocosphaera as opposed to a dual mechanism for
Trichodesmium, the overall effect of elevated pCO
2
might be expected to be smaller in
Crocosphaera in comparison to that in Trichodesmium.
Our data indicate, however, that Crocosphaera WH0401 did fix N
2
during the
beginning of the light period. In high light treatments (≥180 µE m
-2
s
-1
), N
2
-fixation rates
by WH0401 in the present-day and elevated pCO2 treatments remained relatively high
during the early portion of the light cycle in comparison with the low pCO
2
(190 ppm)
83
treatment. These relatively lower N
2
-fixation rates at low pCO
2
are likely due to a
reduced energy supply caused by the exhaustion of glycogen reserves that accumulated
during the preceding light period (Berman Frank et al. 2001, Saito et al. 2011), but may
also result from an increased energy demand for the transport of HCO
3
-
under low pCO
2
conditions. An enhanced capability to fix N
2
during the light period may offer an
advantage to Crocosphaera growing under high pCO
2
and high light, in that N
2
fixation
may benefit from the dual mechanism described above for Trichodesmium.
Although we do not have N
2
-fixation rate data extending beyond the 2
nd
hour of
the light period, based on some of our limited previous data, we assumed that rates
declined rapidly and that additional N
2
fixation beyond this period probably contributed
little to the total daily N
2
fixation. In support of our limited data documenting this diel
trend in N
2
-fixation rates, Mohr et al. (2010a) and Saito et al. (2011) reported that N
2
-
fixation rates by C. watsonii (WH8501) declined rapidly when cultures were exposed to
light. Despite reports of N
2
fixation being highly restricted to the dark period for
WH8501 (Mohr et al. 2010a; Saito et al. 2011), however, our data indicate that N
2
fixation by WH0401 extended into the early portion of the light period.
Gross cellular N
2
-fixation rates in our preliminary CO
2
experiments and CO
2
-light
experiments did not directly track trends in growth rates. Instead, gross:net N
2
-fixation
rate ratios were high in low pCO
2
and low light treatments, suggesting that gross N
2
fixation was in excess of the N demand. Hence, the gross:net N
2
-fixation rate ratio and
growth rates were anti-correlated. Although our gross:net N
2
fixation estimates are
limited to the dark period, this ratio may be different during the early light period when
84
N
2
fixation is coupled with carbon fixation as it is in Trichodesmium, whose gross:net N
2
-
fixation rate ratios were lower, in general, and anti-correlated with light (Garcia et al.
2011). Overall, our data suggest that the incorporation of N
2
into biomass was enhanced
by high light and high pCO
2
when growth rates were maximal.
Thus, based our estimates of gross:net N
2
fixation, high light and high pCO
2
stimulated cellular N retention (Mulholland et al. 2004; Mulholland 2007).
Coincidentally, high gross:net ratios in WH0401 and WH0402 were associated with low
energy conditions in general (low light and low pCO
2
conditions) and may be related to a
compromised phospholipid cell membrane, as is the case for other phytoplankton (Van
Mooy et al. 2006, 2009). Hence, these data suggest that cellular N loss might be reduced
in a high light, high pCO
2
environment. Assuming that these organisms would be
consumed by higher trophic levels in the future, we might expect nitrogen to flow more
efficiently through food webs within the next 100-years, thereby fueling higher secondary
and tertiary production rates. For instance, a high rate of N loss would tend to favor
production within the microbial loop, thereby decreasing the efficiency of N transfer to
higher trophic levels. These higher secondary and tertiary production rates may, in turn,
accelerate carbon drawdown from surface layers of the oceans.
Recently, Mohr et al. (2010b) questioned the validity of the
15
N
2
isotope uptake
method, suggesting that gas solubility issues can lead to large underestimates of actual
net N
2
-fixation rates. Perhaps a better method for estimating net N
2
-fixation rates is by
measuring ∆PN as was done by Kranz et al. (2009). Our estimates indicate that gross:PN
accumulation ratio was close to 1 at non-growth-limiting irradiance. A ratio of 1 seems
85
more intuitively reasonable than the very high estimates of the gross:net N
2
-fixation rate
ratios that we documented (up to 15). But in support of the
15
N
2
isotope uptake method,
15
N
2
-fixation rates and growth rates were strongly correlated in all of our experiments. In
addition,
15
N
2
injections probably equilibrated with non-isotope N
2
gas during our 12-h
incubations. We note that gross N
2
-fixation rates in the CO
2
-light experiment with
WH0401 were amplified in comparison with other experiments because of the
modification of the acetylene assay (see N
2
fixation in the Methods section). These
higher gross N
2
-fixation rates also yielded amplified gross:net ratios in the CO
2
-light
experiment with WH0401 in comparison with other experiments. Because the method
for the acetylene assay technique was not different between light experiments or the
preliminary CO
2
experiments, our best comparisons of the gross:net N
2
-fixation rate
ratios between strains are in Figs 1 and 2.
In summary, growth rates of the large C. watsonii strain WH0402 were higher
than those observed for the smaller WH0401. Our data also imply that WH0402 has a
lower CO
2
requirement (K
1/2
) for growth with respect to CO
2
concentration than
WH0401 at 155 µE m
-2
s
-1
. This conclusion is based on the difference in growth rate
reductions between isolates in response to low pCO
2
in comparison with air treatments
(10-15% for WH0402; ≥40% for WH0401). These data indicate that K
1/2
for growth of
WH0401 with respect to CO
2
is close to 190 ppm pCO
2
, whereas that for WH0402 is
lower. A low energy demand associated with fixing CO
2
may be the reason that
WH0402 has higher growth, N
2
- and CO
2
-fixation rates and a lower CO
2
requirement for
growth than WH0401.
86
While Price et al. (2008) identify different mechanisms by which C. watsonii
(WH8501) acquires carbon, there is no literature describing differences in these
mechanisms between isolates of C. watsonii. Differences in the K
1/2
for growth with
respect to CO
2
might be caused by differences in the efficiency of HCO
3
-
trans-
membrane transport systems, but such attribution of cause and effect must await further
studies with multiple C. watsonii isolates. In further support of an enhanced ability to fix
CO
2
, data reported by Sohm et al. (2011) suggest that extracellular polysaccharide (EPS)
production was considerably higher in the larger strain WH0402 compared to WH0401.
The EPS data suggest that CO
2
fixation is highly excessive with respect to the carbon
demand for growth in WH0402 in comparison with that in WH0401. Unlike those data,
however, we did not find large differences in photosynthetic efficiency of photosystem II
(F
v
/F
m
) between isolates (data not shown).
Based on correlative data presented by Tortell (2000), phytoplankton taxa that
require comparatively high CO
2
concentrations for CO
2
fixation like cyanobacteria also
arose earlier in geologic history, when atmospheric pCO
2
was high relative to present-day
concentrations. Because this correlation applies to clades of phytoplankton rather than
strains of a species, evolutionary differences between WH0401 and WH0402 may have
arisen over shorter time periods. Evolution of an enhanced ability to fix CO
2
might have
developed when pCO
2
concentrations were low relative to present-day concentrations,
such as during glacial maxima (Petit et al. 1999). These relatively short-term adaptations
seem possible considering that they may have been facilitated with transposons rather
than broad-scale gene transfer, deletion or mutation (Zehr et al. 2007). Genetic
87
rearrangement mediated by transposons seems to be the best explanation for
physiological variation among strains of C. watsonii (Zehr et al. 2007). In addition,
strain-specific differences might be caused by biogeochemical differences in their
respective sites of isolation; for instance WH0401 was collected near the Amazon River
plume while WH0402 is likely not adapted to this type of terrestrially-influenced
environment.
88
Chapter 2 Acknowledgements
We thank Dr. Eric Webb for providing isolates of C. watsonii for our experiments and
also for allowing us to use his gas chromatograph for the acetylene reduction assay.
Grant support was provided by NSF OCE 0942379, 0962309, and 1043748 to D.
Hutchins, NSF OCE 0850730 to F.-X. Fu, and NSF OCE 0722395 to M. Mulholland.
89
Chapter 2 References
Alley, R.B., Berntsen, T., Bindoff, N.L., Chen, Z. and others. Summary for
policymakers. In: Solomon S, Qin D, Manning M, Chen Z and others (Eds.) Climate
change 2007: The physical science basis. Contribution of Working Group I to the fourth
assessment report of the Intergovernmental Panel on Climate Change. Cambridge
University Press, Cambridge and New York. (2007).
Badger, M. R., Price, G. D., Long, B. M. & Woodger, F. J. 2006. The environmental
plasticity and ecological genomics of the cyanobacterial CO
2
concentrating mechanism.
J. Exp. Bot. 57:249–265.
Barcelos e Ramos, J., Biswas, H., Schulz, K. G., LaRoche, J. & Riebesell, U. 2007.
Effect of rising atmospheric carbon dioxide on the marine nitrogen fixer Trichodesmium.
Glob. Biogeochem. Cycles 21:GB2028.
Behrenfeld, M.J., O’Mally, R. T., Siegel, D.A., McClain, C.R., Sarmiento, J.L., Feldman,
G.C., Milligan, A.J., Falkowski, P.G., Letelier, R.M., Boss, E.S. 2006. Climate-driven
trends in contemporary ocean productivity. Nature 444:752-755.
Berman-Frank, I., Lundgren, P., Chen, Y-B., Kupper, H., Kolber, Z., Bergman, B. &
Falkowski, P. 2001. Segregation of nitrogen fixation and oxygenic photosynthesis in the
marine cyanobacterium Trichodesmium. Science 294:1534-1537
Boyd, P.W., Strzepek, R., Fu, F., Hutchins, D.A. 2010. Environmental control of open-
ocean phytoplankton groups: now and in the future. Limnol. Oceanogr. 55:1353-1376.
Breitbarth, E., Mills, M. M., Friedrichs, G. & LaRoche, J. 2004. The Bunsen gas
solubility coefficient of ethylene as a function of temperature and salinity and its
importance for nitrogen fixation assays. Limnol. Oceanogr.: Methods 2:282-288.
Capone, D. G. 1993. Determination of nitrogenase activity in aquatic samples using the
acetylene reduction procedure. In: Kemp, P. F., J. J. Cole, B. F. Sherr, E. B. Sherr (eds).
Handbook of methods in aquatic microbial ecology. Lewis Publishers, Boca Raton, FL, p
621–631.
Capone, D. G. 2008. The marine nitrogen cycle. Microbe 3:186-192.
Chen, Y. B., Zehr, J. P. & Mellon, M. 1996. Growth and nitrogen fixation of the
diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp IMS 101
in defined media: Evidence for a circadian rhythm. J. Phycol. 32:916-923.
90
Church, M. J., Björkman, K. M., Karl, D. M., Saito, M. A. & Zehr, J. P. 2008. Regional
distributions of nitrogen-fixing bacteria in the Pacific Ocean. 53:63-77.
Deutsch, C., Sarmiento, J. L., Sigman, D. M., Gruber, N. & Dunne, J. P. 2007. Spatial
coupling of nitrogen inputs and losses in the ocean. Nature 445:163-167.
Fu, F.-X., Mulholland, M. R., Garcia, N. S., Beck, A., Bernhardt, P. W., Warner, M. E.,
Sanudo-Wilhelmy, S. A. & Hutchins, D. A. 2008. Interactions between changing pCO
2
,
N
2
fixation, and Fe limitation in the marine unicellular cyanobacterium Crocosphaera.
Limnol. Oceanogr. 53:2472-2484.
Garcia, N. S., Fu, F-X, Breene, C. L., Bernhardt, P. W., Mulholland, M. R. Sohm, J. A. &
Hutchins, D. A. 2011. Interactive effects of irradiance and CO
2
on CO
2
- and N
2
fixation
in the diazotroph Trichodesmium erythraeum (Cyanobacteria). J. Phycol. 47:1292-1303.
Hutchins, D. A., Fu, F. X., Zhang, Y., Warner, M. E., Feng, Y., Portune, K., Bernhardt,
P. W. & Mulholland, M. R. 2007. CO
2
control of Trichodesmium N
2
-fixation,
photosynthesis, growth rates, and elemental ratios: Implications for past, present, and
future ocean biogeochemistry. Limnol. Oceanogr. 52:1293-1304.
Hutchins, D. A., Mulholland, M. R., & Fu, F.-X. 2009. Nutrient cycles and marine
microbes in a CO
2
enriched ocean. Oceanography 22:128-145.
Kranz, S. A., Sultemeyer, D., Richter, K. U., & Rost, B. 2009. Carbon acquisition by
Trichodesmium: The effect of pCO
2
and diurnal changes. Limnol. Oceanogr. 54:548-559.
Kranz, S. A., Levitan, O., Richter, K-U., Prášil, O., Berman-Frank, I., & Rost, B. 2010.
Combined effects of CO
2
and light on the N
2
-fixing cyanobacterium Trichodesmium
IMS101: Physiological responses. Plant Physiol. 154:334-345.
Levitan, O., Rosenberg, G., Šetlík, I., Setlikova, E., Grigel, J., Klepetar, J., Prasil, O. &
Berman-Frank, I. 2007. Elevated CO
2
enhances nitrogen fixation and growth in the
marine cyanobacterium Trichodesmium. Glob. Change Biol. 13:531-538.
Levitan, O., Kranz, S. A., Spungin, D., Prášil, O., Rost, B. & Berman-Frank, I. 2010.
Combined effects of CO
2
and light on the N
2
-fixing cyanobacterium Trichodesmium
IMS101: A mechanistic view. Plant Physiol. 154:346-356.
Moisander, P. H., Beinart, R. A., Hewson, I., White, A. E., Johnson, K. S., Carlson, C.
A., Montoya, J. P, & Zehr, J. P. 2010. Unicellular cyanobacterial distributions broaden
the oceanic N
2
fixation domain. Science. 327:1512-1514.
91
Montoya, J. P., Holl, C. M., Zehr J. P., Hansen, A., Villareal, T. A. & Capone, D. G.
2004. High rates of N
2
fixation by unicellular diazotrophs in the oligotrophic Pacific
Ocean. 430:1027-1031.
Morel, F. M. M., Rueter, J. G., Anderson, D. M. and Guillard, R. R. L. 1979. Aquil -
chemically defined phytoplankton culture-medium for trace-metal studies. J. Phycol.
15:135-141.
Mohr, W., Intermaggio, M.P. & J. LaRoche. 2010a. Diel rhythm of nitrogen and carbon
metabolism in the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii
WH8501. Environ. Microbiol. 12:412-421.
Mohr, W., Großkopf, T., Wallace, D.W.R. & J. LaRoche. 2010b. Methodological
underestimation of oceanic nitrogen fixation rates. PLoS ONE. 5:e12583.
Monod, J. 1949. The growth of bacterial cultures. Annu. Rev. Microbiol. 3:371-394.
Mulholland, M. R. 2007. The fate of nitrogen fixed by diazotrophs in the ocean.
Biogeosciences Discuss. 4:37-51.
Mulholland, M. R. and Bernhardt, P. W. 2005. The effect of growth rate, phosphorus
concentration and temperature on N
2
-fixation, carbon fixation, and nitrogen release in
continuous cultures of Trichodesmium IMS101. Limnol. Oceangr. 50:839-849.
Mulholland, M. R., Bronk, D. A. & Capone, D. G. 2004. N
2
fixation and regeneration of
NH
4
+
and dissolved organic N by Trichodesmium IMS101. Aquatic Microb. Ecol. 37:85-
94.
Mulholland, M. R. and Bernhardt, P. W. 2005. The effect of growth rate, phosphorus
concentration and temperature on N
2
-fixation, carbon fixation, and nitrogen release in
continuous cultures of Trichodesmium IMS101. Limnol. Oceangr. 50:839-849.
Mulholland, M.R., Bernhardt, P.W. Blanco-Garcia, J.L., Manino, A., Hyde, K.,
Mondragon, E. Turk, K., Moisander, P.H. & Zehr, J.P. 2012. Rates of dinitrogen fixation
and the abundance of diazotrophs in North American coastal waters between Cape
Hatteras and Georges Bank. Limnol. Oceanogr. In press.
Petit, J.R., Jouzel, J., Raynaud, D., Barkov, N.I., Barnola, J.-M., Basile, I., Bender, M.,
Chapellaz, J., Davis, M., Delaygue, G., Delmott, M., Kotlyakov, V.M., Legrand, M.,
Lipenkov, V.Y., Lorius, C., Pépin, L., Ritz, C., Saltzman, E. & Stievenard, M. 1999.
Climate and atmospheric history of the past 420,000 years from the Vostok ice core,
Antarctica. Nature 399:429-436.
92
Price, G. D., Badger, M. R., Woodger, F. J. & Long B. M. 2008. Advances in
understanding the cyanobacterial CO
2
-concentrating-mechanism (CCM): functional
components, Ci transporters, diversity, genetic regulation and prospects for engineering
into plants. J. Exp. Bot. 59:1441–1461.
Saito, M.A., Bertrand, E.M., Dutkiewics, S., Bulygen, V.V., Moran, D.M., Montiero,
F.M., Follows, M.J., Valois, F.W. & J.B. Waterbury. 2011. Iron conservation by
reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii.
Proc. Natl. Acad. Sci. 108:2184-2189.
Samuelsson, G. & Öquist, G. 1977. A method for studying photosynthetic capacities of
unicellular algae based on in vivo chlorophyll fluorescence. Physiologia Plantarum.
40:315-319.
Sarmiento, J.L., Slater, R., Barber, R., Bopp, L., Doney, S.C., Hirst, A.C., Kleypas, J.,
Matear, R., Mikolajewics, U., Monfrey, P., Soldatov, V., Spall, S.A., & R. Stouffer.
2004. Response of ocean systems to climate warming. Global Biogeochem. Cy. GB3003.
doi:10.1029/2003GB002134.
Sohm, J.A., Edwards, B.R., Wilson, B.G., and E. A. Webb. 2011. Constitutive
extracellular polysaccharide (EPS) production by specific isolates of Crocosphaera
watsonii. Frontiers in Microbiology. 229. doi: 10.3389/fmicb.2011.00229
Tortell, P. D. 2000. Evolutionary and ecological perspectives on carbon acquisition in
phytoplankton. Limnol. Oceanogr. 45:744-750.
Van Mooy, B.A.S., Rocap, G. Fredericks, H.F., Evans, C.T., Devol, A.H. 2006.
Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in
oligotrophic marine environments. PNAS. 103: 8607-8612.
Van Mooy, B.S., Fredericks, H.F., Pedler, B.E. Dyhrman, S.T., Karl, D.M., Koblížek, M.,
Lomas, M.W., Mincer, T.J., Moore, L.R., Moutin, T., Rappé, M.S., Webb, E.A. 2009.
Phytoplankton in the ocean use non-phospholipids in response to phosphorus scarcity.
Nature. 458: 69-72.
Webb, E.A., Ehrenreich, I.M., Brown, S.L., Valois, F.W. & J. B. Waterbury. 2009.
Phenotypic and genotypic characterization of multiple strains of the diazotrophic
cyanobacterium Crocosphaera watsonii, isolated from the open ocean. Environmental
Microbiology. 11-338-348.
Zehr, J. P., Waterbury, J. B, Turner, P. J., Montoya, J. P., Omoregie, E., Steward, G. F.,
Hansen, A., & Karl, D. M. 2001. Unicellular cyanobacteria fix N
2
in the subtropical
North Pacific Ocean. Nature 412: 635-638.
93
Zehr, J. P., Bench, S. R., Mondragon, E. A., McCarren, J., & DeLong, E. F. 2007. Low
genomic diversity in tropical oceanic N
2
-fixing cyanobacteria. Proc. Nat. Acad. Sci.
1780-17812.
94
Chapter 2 Table 1.
95
Chapter 2 Table 2.
96
Chapter 2 Figures
Chapter 2 Figure 1. Specific growth rates (d
-1
) (a), cell diameter (µm) (b), carbon-
specific CO
2
-fixation rates (c), N-specific gross N
2
-fixation rates (d), N-specific net N
2
-
fixation rates (e), and gross:net N
2
-fixation rate ratios (f) of Crocosphaera watsonii,
isolates WH0401 and WH0402, in response to irradiance (25-300 µmol quanta m
-2
s
-1
).
Data correspond to experiments 1 and 2 in Table 1. Isolates were grown with a semi-
continuous culturing method. Open symbols are WH0401; closed symbols are WH0402.
Error bars are the standard errors on the means of three experimental replicates.
Chapter 2 Figure 1.
97
Chapter 2 Figure 2. Specific growth rates and gross:net N
2
-fixation rate ratios (a,b),
cellular gross N
2
- and net N
2
-fixation rates (c,d) of Crocosphaera watsonii, isolates
WH0401 and WH0402, under different pCO
2
levels. Data correspond to experiments 3
and 4 in Table 1. Cultures were grown with a semi-continuous culturing method at 155
mmol quanta m
-2
s
-1
. Error bars are the standard errors on the means of three
experimental replicates.
Chapter 2 Figure 2.
98
Chapter 2 Figure 3. Cellular CO
2
-fixation rates (a), particulate nitrogen (PN)
accumulation rates (b) and gross: PN accumulation rate ratios (c) of Crocosphaera
watsonii, isolates WH0401 and WH0402, under different pCO
2
levels. Data correspond
to experiments 3 and 4 in Table 1. Cultures were grown with a semi-continuous culturing
method at 155 mmol quanta m
-2
s
-1
. Error bars are the standard errors on the means of
three experimental replicates.
Chapter 2 Figure 3.
99
Chapter 2 Figure 4. Specific growth rates (a,b) and cellular CO
2
-fixation rates (c,d) of
Crocosphaera watsonii, in semi-continuous cultures grown under a range irradiance and
different pCO
2
levels. WH0401 and WH0402 were grown under present-day and elevated
pCO
2
levels and WH0401 was also grown under 190 ppm pCO
2
. Data correspond to
experiments 5 and 6 in Table 1. Open symbols are 750 or 761 ppm pCO
2
treatments;
gray symbols are air treatments; closed symbols are 190 ppm pCO
2
treatments. Error bars
are the standard errors on the means of three experimental replicates.
Chapter 2 Figure 4.
100
Chapter 2 Figure 5.
101
Chapter 2 Figure 5. Cellular gross N
2
-fixation rates (a,b), cellular net N
2
-fixation rates
(c,d), calculated cellular particulate nitrogen (PN) accumulation rates (e,f), N-specific
gross N
2
-fixation rates (g,h), N-specific net N
2
-fixation rates (i, j) in semi-continuous
cultures of Crocosphaera watsonii, isolates WH0401 and WH0402, as a function of
pCO
2
and irradiance. WH0401 and WH0402 were grown under present-day and elevated
pCO
2
levels and WH0401 was also grown under 190 ppm pCO
2
. Data correspond to
experiments 5 and 6 in Table 1. Open symbols are 750 or 761 ppm pCO
2
treatments;
gray symbols are air treatments; closed symbols are 190 ppm pCO
2
treatments. Error bars
are the standard errors on the means of three experimental replicates.
Chapter 2 Figure 5 Caption.
102
Chapter 2 Figure 6. Gross:net N
2
-fixation rate ratios (a,b) and gross:net PN accumulation
ratios (c,d) in semi-continuous cultures of Crocosphaera watsonii, isolates WH0401 and
WH0402, as a function of pCO
2
and irradiance. WH0401 and WH0402 were grown
under present-day and elevated pCO
2
levels and WH0401 was also grown under 190 ppm
pCO
2
. Data correspond to experiments 5 and 6 in Table 1. Open symbols are 750 or 761
ppm pCO
2
treatments; gray symbols are air treatments; closed symbols are 190 ppm
pCO
2
treatments. Error bars are the standard errors on the means of three experimental
replicates.
Chapter 2 Figure 6.
103
Chapter 2 Figure 7. Changes in gross N
2
-fixation rates during the dark (shaded area) and
early light period (non-shaded area) in Crocosphaera watsonii strain WH0401 grown at
irradiances of 300 (a), 180 (b) and 100 (c) µmol quanta m
-2
s
-1
. Data correspond to
experiment 5 in Table 1. Open symbols are 761 ppm pCO
2
treatments; gray symbols are
air treatments; closed symbols are 190 ppm pCO
2
treatments. Error bars are the standard
errors on the means of three experimental replicates.
Chapter 2 Figure 7.
104
Chapter 3
COLIMITATION INTERACTIONS BETWEEN PHOSPHORUS, LIGHT AND
CO
2
IN THE UNICELLULAR PHOTOSYNTHETIC DIAZOTROPH
CROCOSPHAERA WATSONII
Nathan S. Garcia, Fei-Xue Fu, Elizabeth K. Yu, David A. Hutchins
3
Department of Biological Sciences, University of Southern California, Los Angeles, CA
90089, USA
3
Corresponding author; dahutch@usc.edu
105
Chapter 3 Abstract
Along with rising atmospheric partial pressure of carbon dioxide (pCO
2
), mean mixed
layer irradiance and phosphorus (P) concentrations in mixed layers of the ocean are
expected to change within the next 100 years due to a warmer, more stratified ocean
surface. Future trends in these three variables could have large implications for
biogeochemically-critical N
2
-fixing cyanobacteria. Here we document interactive effects
of P (0.1 - 4.0 µM), irradiance (40 and 150 µmol quanta m
-2
s
-1
) and pCO
2
(190 and 800
ppm) on growth, CO
2
- and dinitrogen (N
2
)-fixation rates of the unicellular N
2
-fixing
cyanobacterium Crocosphaera watsonii (WH0003) using a large cell strain (4.5-6.5 µM
diameter) isolated from the Pacific Ocean near Hawaii. The critical threshold (T
cr
) and
half-saturation constant (K
1/2
) for growth with respect to P were reduced by 50 nM under
elevated pCO
2
(800 ppm) when compared with low pCO
2
(190 ppm). This trend was
consistent in low-light and high-light cultures. We also documented a 100-nM increase
in T
cr
and K
1/2
for growth with respect to P under high light in comparison with low-light
cultures. We speculate that low T
cr
and K
1/2
values under high pCO
2
resulted from a
lower energy demand associated with carbon concentrating mechanisms, in comparison
with low-pCO
2
cultures. Our results imply that changing concentrations of P, CO
2
and
light, will have both positive and negative effects on growth, CO
2
- and N
2
-fixation rates
by unicellular oxygenic diazotrophs like C. watsonii within the next 100 years. We
expect that the net effect on C. watsonii might be more positive in the Pacific Ocean
relative to the Atlantic Ocean.
106
Chapter 3 Introduction
Within the past five years, new attention has focused on the effects of global
change on marine dinitrogen (N
2
) fixation. In particular, three studies initiated this
interest by documenting increased N
2
-fixation rates by Trichodesmium erythraeum in
response to elevated partial pressures of carbon dioxide (pCO
2
) (Hutchins et al. 2007;
Levitan et al. 2007 and Barcelos e Ramos et al. 2007). In one of these studies, Hutchins
et al. (2007) examined the combined effects of pCO
2
, temperature and orthophosphate
concentration (PO
4
3-
) on N
2
-fixation rates by two strains of T. erythraeum. This type of
study is particularly important because it aims to answer questions about how multiple
environmental factors might change and interact to affect ocean biogeochemical cycles.
That study concluded that pCO
2
limitation of N
2
-fixation rates by T. erythraeum was
independent of PO
4
3-
concentrations, because the relative magnitude of elevated pCO
2
effects on growth and N
2
-fixation rates was nearly identical in P-limited and P-replete
cultures. This finding suggests that decreases in oceanic P concentrations, which might
accompany future changes in surface ocean temperatures and stratification (Hutchins et
al. 2009) would not affect the seemingly large impact of elevated pCO
2
on oceanic N
2
fixation by Trichodesmium.
More recent studies indicate that the light environment can moderate the effect of
elevated pCO
2
on N
2
-fixation rates by T. erythraeum; the stimulatory effect of elevated
pCO
2
is reduced under high light relative to lower light intensities (Kranz et al. 2010;
Garcia et al. 2011). While it is clear that interactive effects of multiple environmental
factors must be considered for Trichodesmium, these oceanic diazotrophs may contribute
107
less than half of the world’s marine biological N
2
fixation. Unicellular N
2
fixers like
Crocosphaera watsonii are also now recognized as major contributors to marine
biological N
2
-fixation (Zehr et al. 2001; Montoya et al. 2004; Moisander et al. 2010), and
we need a clearer understanding of how these organisms will respond to global change as
well (Fu et al. 2008, Garcia et al. in press).
Two studies suggest that elevated pCO
2
has a positive effect on N
2
-fixation rates
by C. watsonii, and that light and iron interact with CO
2
in different ways to moderate
this effect (Fu et al. 2008; Garcia et al. in press). The influence of elevated pCO
2
on N
2
-
fixation rates is thought to be less direct for Crocosphaera than it is for Trichodesmium,
however, because of their contrasting N
2
-fixation strategies (Garcia et al. in press). This
work suggests that the temporal separation of CO
2
- and N
2
fixation by Crocosphaera has
different physiological and biogeochemical consequences in terms of responses to
interacting global change variables, when compared to Trichodesmium which fixes both
CO
2
and N
2
simultaneously. Here, we examine how changes in the availability of P, a
key-limiting nutrient for diazotrophs, can affect this light/CO
2
interaction in a
Crocosphaera watsonii strain isolated from the subtropical Pacific Ocean.
108
Chapter 3 Methods
We conducted a light experiment and a P-light-CO
2
experiment with
Crocosphaera watsonii (WH0003) that was isolated near station ALOHA in the North
Pacific Ocean near Hawaii (22°45’N, 158°00’W). Experimental cultures were grown
with a semi-continuous culturing method (Garcia et al. 2011) at 28°C in an autoclave-
sterilized artificial seawater medium following the recipe of Chen et al. (1996). Nutrients
were added in concentrations equivalent to the AQUIL recipe (Morel et al. 1979) except
for NO
3
-
. The PO
4
3-
concentration was altered in the P-light-CO
2
manipulation
experiment.
In the light experiment, triplicate cultures were grown in 800 mL polystyrene
flasks under 5 irradiances (18, 40, 100, 180, 300 µmol quanta m
-2
s
-1
) and diluted every
2-3 days to 10-20 x 10
3
cells mL
-1
. The PO
4
-3
concentration was measured in previous
experiments that had a very similar design and was never recorded below 15 µM, before
or after dilutions. In the P-light-CO
2
experiment, triplicate cultures were diluted with
medium that contained treatment concentrations of PO
4
3-
ranging from 0.1 - 4.0 µM.
Cultures were diluted every two days to 5x10
3
cells mL
-1
. A low cell biomass was
necessary to control CO
2
concentrations in cultures and a consistent dilution period
reduced variations in growth rates between dilutions. Cultures were grown in 1-L
polycarbonate bottles at 40 or 150 µmol quanta m
-2
s
-1
and bubbled with 800 ppm or 190
ppm pCO
2
pre-mixed air supplied and certified by Gilmore Liquid Air Company. Culture
pH was measured with a pH meter using the NBS seawater scale (model: Orion 5 star
Thermo Scientific, Beverly, MA, USA). For the P-light-CO
2
experiment seawater was
109
pre-bubbled and equilibrated with treatment concentrations of pCO
2
before measuring pH
and adding nutrients.
In both experiments, steady state growth rates were calculated after cultures were
acclimated to treatment conditions for 7-10 generations and cultures were then terminally
sampled for N
2
- and CO
2
-fixation rates. In the P-light-CO
2
experiment, cultures were
sampled 24 hours after the last dilution when we also measured
33
P-uptake rates and
elemental composition. Light was supplied on a 12:12 light:dark cycle with cool white
fluorescent bulbs.
Nitrogen-fixation rates were determined with the acetylene reduction method as
described in Garcia et al. (submitted). Briefly, duplicate 50 mL culture samples were
collected from experimental replicates and 4 mL of acetylene was injected into 30 mL
headspace at the beginning of the dark period of the light cycle. Samples were gently
agitated to equilibrate gas concentrations in the headspace and culture samples after
injecting acetylene and before measuring ethylene concentrations. We used a Bunsen
coefficient for ethylene of 0.082 (Breitbarth et al. 2004) and an ethylene production:N
2
fixation rate ratio of 3:1. We calculated N
2
-fixation rates over 14 h to account for 2 h of
N
2
fixation during the early portion of the light cycle, as documented for C. watsonii in
Garcia et al. (submitted).
We determined CO
2
-fixation rates using liquid scintillation counting according to
the method described in Garcia et al. (2011). Briefly, total CO
2
concentrations (TCO
2
)
were multiplied by the ratio of radioactivity of cellular incorporation of
14
C during 24 h
to the total radioactivity of H
14
CO
3
-
(see Table 1 for TCO
2
measurements). For CO
2
-
110
fixation rates in our light experiment, we used a mean concentration of 2061 µM TCO
2
that was measured in previous experiments with C. watsonii (Garcia et al. in press). For
CO
2
-fixation rates in our P-light-CO
2
experiment, we pooled ~25 mL from each of 3
treatment replicates into one sample for TCO
2
measurements. Samples were preserved
with 0.05% mercuric chloride in glass bottles without headspace and were measured with
a carbon coulometer as described in Garcia et al. (2011).
Phosphorus uptake rates were determined with radioactive
33
PO
4
3-
over 24 hrs
similarly to the method used for H
14
CO
3
-
uptake described here. Because
33
PO
4
3-
was
added at only trace concentrations, we assumed that the total PO
4
3-
concentration was
equal to treatment concentrations.
Near the end of the light period (9
th
-11
th
hour), we filtered samples for cellular N
and C (120 mL) and P content (50 mL) from each replicate onto combusted (450°C, 4 h)
Whatman GF/F filters and measured them following the description by Fu et al. (2005).
For C and N measurements, filtered samples were dried and pelletized before analysis
with an elemental analyzer (Costech Instruments, model 4010). For cellular P content,
filtered samples were rinsed 3 times with 2 mL 0.017 M Na
2
SO
4
and placed in 20 mL
glass scintillation vials with 2 mL 0.017 MgSO
4
, which was evaporated at ~80°C over a
few days. Filters were combusted at 450°C for 2 hrs to release P from organic
compounds. After cooling, filters were reheated to 80°C along with 5 mL 0.2 M HCl for
30 minutes and phosphate concentrations were estimated spectrophotometrically with the
colorimetric assay described by Lebo and Sharp (1992).
111
Cells were counted microscopically with a hemocytometer and cell diameters of
12 cells were determined in our light experiment with an ocular micrometer. Growth
rates were calculated by estimating culture cell density between dilutions as in Garcia et
al. (submitted). Growth rates, CO
2
- and N
2
-fixation rates were fitted to the linear
hyperbolic function of PO
4
3-
concentrations as described in Garcia et al. (submitted) for
the P-light-CO
2
experiment. For the light experiment the hyperbolic function of CO
2
- and
N
2
-fixation rates were fitted to irradiance without including the rates measured at 100
µmol quanta m
-2
s
-1
as we suspect that irradiance may have changed in this treatment just
prior to sampling due to other ongoing laboratory experiments in the incubator. We used
the analysis of variance statistical tests (JMP v.4) combined with a Tukey analysis of
multiple comparisons to determine statistical differences (p<0.05) between treatments.
112
Chapter 3 Results
Half-saturation constants (K
1/2
) for growth and N
2
-fixation rates of C. watsonii
(WH0003) with respect to light were relatively close to each other (76 and 94 µmol
quanta m
-2
s
-1
, Table 2). Although the critical threshold (T
cr
, the concentration that yields
a rate of zero) for light density was identical for growth, N
2
-fixation and CO
2
-fixation
rates, the K
1/2
for CO
2
fixation was roughly twice as high as those for N
2
-fixation and
growth rates (Table 2, Figure 1a,b,c). Two-way ANOVA indicated that cell diameter
increased as a function of increasing light (F
4,110
=31, p<0.0001) and similar to results
reported by Mohr et al. (2010), cell diameter increased during the light period (F
1,110
=8.7,
p<0.01) and was dependent on the cellular C content (Fig. 1d).
In our P-light-CO
2
experiment, culture pH and TCO
2
concentrations in treatments
of pCO
2
were close to ranges documented in previous culture studies with C. watsonii
grown at 190 and 750 ppm pCO
2
(Table 1; Garcia et al. in press). Growth, N
2
-fixation
and CO
2
-fixation rates at 40 and 150 µmol quanta m
-2
s
-1
were similar to rates in cultures
incubated at similar irradiance levels in our light experiment (Figures 1, 2 and 3).
Functional response curves with respect to light suggest that growth and N
2
-fixation rates
were not light limited at 150 µmol quanta m
-2
s
-1
in our P-light-CO
2
experiment, despite
the fact that CO
2
-fixation rates at 150 µmol m
-2
s
-1
were under saturated with light.
Overall, K
1/2
values for growth with respect to PO
4
3-
were close to previously
reported values for large unicellular diazotrophs (Falcon et al. 2005) and varied as a
function of light and pCO
2
(Table 3). The K
1/2
and T
cr
for growth and N
2
-fixation rates
with respect to PO
4
3-
increased by approximately 0.1 µM PO
4
3-
as a function of
113
increasing irradiance and by approximately 0.05 µM as a function of decreasing pCO
2
.
Under high light (150 µmol quanta m
-2
s
-1
), functional response parameters (K
1,2
and T
cr
)
for N
2
-fixation rates were nearly identical to those for growth rates and relative changes
in maximum rates between pCO
2
treatments were similar for N
2
fixation and growth
rates. Under low light, there was a large difference in maximum growth rates between
the 190 and 800 ppm CO
2
treatments, relative to high light treatments (a 130% increase at
40 µmol quanta m
-2
s
-1
compared to 60% increase at 150 µmol quanta m
-2
s
-1
). Two-way
ANOVA indicated that light and pCO
2
had a significant positive interactive effect on N
2
-
fixation rates in P-replete treatments (4.0 µM PO
4
3-
; F
1,8
=7.8, p=0.02); light had a
significant positive effect on N
2
-fixation rates (F
1,8
=72, p<0.001). Elevated pCO
2
did not
have a significant effect on N
2
fixation under low light (F
1.8
=0.04, p=0.85) but had a
significant positive effect under high light (F
1,8
=17, p=0.003).
In the low-light, low-pCO
2
treatment at 0.15 µM PO
4
3-
growth was not
sustainable, despite having positive N
2
-fixation rates. These data support previous
findings that the gross:net N
2
-fixation rate ratio (a proxy for cellular retention of fixed N;
see Mulholland et al., 2004 and Mulholland 2007) was lowest under high light and high
pCO
2
in C. watsonii, implying that these two conditions interactively favor cell retention
of fixed N (Garcia et al. in press).
As a result of the shift in T
cr
for growth with respect to PO
4
3-
, the effect of
elevated pCO
2
was much stronger in low P treatments than in P-replete conditions. In
high-light treatments, growth rates and N
2
-fixation increased by 240% and 540%,
respectively as a function of pCO
2
in the 0.25 µM PO
4
3-
treatments (Figs. 2a and 3c). In
114
the 4.0-µM PO
4
3-
treatments however, growth and N
2
-fixation rates only increased by
64% and 58%, respectively, in response to elevated pCO
2
. Under low light, the trend was
similar; the effect of elevated pCO
2
on growth and N
2
-fixation rates was moderated by
PO
4
3-
concentration (Figs. 2b, 3d). At 40-µmol quanta m
-2
s
-1
, elevated pCO
2
yielded a
90% increase in N
2
-fixation rates in the 0.15-µM PO
4
3-
treatments but only an 8%
increase at 4.0-µM PO
4
3-
(Fig. 3c).
Effects of pCO
2
and PO
4
3-
on kinetic constants of CO
2
-fixation rates under high
light were similar to those for growth and N
2
-fixation rates (Table 3). We could not
determine meaningful kinetic parameters for our low light treatments however, because
CO
2
-fixation rates (Fig. 3b) did not follow the same trends as growth rates (Fig. 2a) and
N
2
-fixation rates (Fig. 3d) under low P conditions. In P-replete treatments, the
combination of high light and high pCO
2
had a strong positive interactive effect on CO
2
-
fixation rates (F
3,8
=9.6, p=0.01); in the 4.0-µM PO
4
3-
treatment, CO
2
-fixation rates were
not significantly different between pCO
2
treatments at low light (Fig. 3b, F
1,8
=1.0,
p=0.34) but were at high light (Fig. 3a, F
1,8
=9.9, p=0.01). Similarly, high light and high
pCO
2
had a strong positive interactive effect on N
2
-fixation rates under P-replete
conditions (F
3,7
=6.1, p=0.04); in the 4.0-µM PO
4
3-
treatment, N
2
-fixation rates were not
significantly different between pCO
2
treatments at low light (Fig. 3d, F
1,7
=0.3, p=0.87)
but were at high light (Fig. 3c, F
1,7
=15, p=0.006; Fig. 3).
In P-replete cultures (4.0 µM treatments), P-uptake rates approximated trends in
growth rates (Fig. 4 a,b). In low-P cultures (≤ 0.4 µM), P-uptake rates were lower under
high light in comparison with low light treatments (Fig. 4a). In high-light, low-P
115
cultures, elevated pCO
2
had a significant positive effect on P-uptake rates (p<0.05; Fig.
4a), following trends in growth rates and as a result, the P-uptake:growth rate ratio did
not vary widely in high light cultures (Fig. 4b). In low-light, low-P cultures P-uptake
rates were not different between pCO
2
treatments (p<0.05; Fig. 4a) and as a consequence
of the differential shift in P-uptake rates relative to growth rates as a function of pCO
2
and light (Fig. 4b), mean cellular P content (fmol P cell
-1
) was significantly higher in
low-light cultures than in high-light cultures and was highest in the low-light, low-pCO
2
treatment (Fig. 4c).
In P-replete cultures (4.0 µM PO
4
3-
), the ratio of cellular P content:CO
2
fixation
rate and cellular P content:N
2
fixation rate were significantly higher in low light
treatments relative to high light treatments (F
1,8
>11.8, p<0.01; Fig 5 a,b). Overall, pCO
2
had a significant positive effect on the cellular P content:N
2
fixation rate ratio (F
1,8
=4.9,
p=0.05; Fig. 5b). Although mean ratios of cellular P content:CO
2
fixation were higher in
low pCO
2
treatments relative to high pCO
2
treatments within each light treatment, the
effect of pCO
2
was not significant (F
1,8
=1.3, p=0.28, Fig. 5a).
116
Chapter 3 Discussion
Our main finding is that T
cr
and K
1/2
for growth, CO
2
- and N
2
-fixation rates with
respect to PO
4
3-
by C. watsonii (WH0003) were strongly affected by pCO
2
. The T
cr
and
K
1/2
values shifted 50 nM between cultures growing under the last glacial maximum level
of atmospheric CO
2
, and the year 2100 predicted concentration (Alley et al. 2007-IPCC;
Royal Society 2005; Petit et al. 1999). As a result of this shift, we documented an 8-fold
increase in the effect of elevated pCO
2
on N
2
-fixation rates in low-P-acclimated cultures
in comparison with P-replete cultures. These effects of elevated pCO
2
were similar under
both growth-limiting irradiance (40 µmol quanta m
-2
s
-1
) and higher light intensity (150
µmol quanta m
-2
s
-1
).
These results indicate that the effect of elevated pCO
2
on growth, CO
2
- and N
2
fixation by C. watsonii is dependent on the availability of P. This finding is in contrast to
trends documented for Trichodesmium. Hutchins et al. (2007) reported that P and CO
2
affected growth and N
2
-fixation rates by T. erythraeum (IMS101) independently of each
other; relative percent changes in N
2
fixation and other rate parameters due to elevated
pCO
2
were similar in P-limited and P-replete cultures. The reason for this difference
between Trichodesmium and Crocosphaera in the interdependency of P and CO
2
concentrations is not presently known, but may be caused by the difference in N
2
-fixation
strategies used by these two very different oceanic diazotrophs (i.e. temporal vs. spatial
separation of N
2
fixation from CO
2
fixation; Berman-Frank et al. 2007).
We speculate that the increase in the T
cr
for growth associated with low pCO
2
is
caused by an increased energy demand by carbon concentrating mechanisms. In low-
117
pCO
2
-acclimated cultures, the energy used by carbon concentrating mechanisms (CCMs)
to import bicarbonate (HCO
3
-
) and convert CO
2
to HCO
3
-
intracellularly is thought to be
high relative to that in high-pCO
2
cultures (Badger et al. 2006; Price et al. 2008; Kranz et
al. 2011). A high-energy demand by CCMs leaves lower energy reserves available to
fuel other high energy demanding processes like cell division, CO
2
- and N
2
fixation. In
Crocosphaera, low CO
2
-fixation rates probably lead to low glycogen production and
storage during the light period (Garcia et al. in press), which would later lead to a
reduced N
2
-fixation rate during the dark period (Saito et al. 2011).
In high-light and low-pCO
2
treatments, this type of energetic deficit is also likely
responsible for the low P-uptake rates in comparison with high-light, high-pCO
2
treatments. Dyhrman and Haley (2006) identified genes for an inducible high-affinity P
transport system (PstS) in C. watsonii that is likely an energetically demanding
mechanism (Scanlan et al. 1993; Donald et al. 1997). Thus, we propose that a reduced
ATP pool available for these high energy-demanding processes directly causes the high
T
cr
for growth, N
2
- and CO
2
-fixation rates under low pCO
2
. This interpretation implies
that the T
cr
for growth is determined by an interactive colimitation relationship between
N, C, and P.
The apparent effects of this low-pCO
2
-induced energy limitation are different
under low irradiance. At low light, CO
2
-fixation rates were not different between pCO
2
treatments, and are might be responsible for the minor effect of elevated pCO
2
on N
2
-
fixation rates in comparison with that at higher irradiance. In support of these findings,
light and pCO
2
had a strong positive interactive effect on CO
2
-fixation rates in T.
118
erythraeum (Garcia et al. 2011) and a large-cell isolate of C. watsonii isolated from the
Atlantic Ocean (Garcia et al. in press); in these two experiments, elevated pCO
2
had a
strong effect on CO
2
-fixation rates under high light, but had little or no effect under low
light.
Despite this lack of difference in CO
2
- and N
2
-fixation rates between pCO
2
treatments at low light, in P-replete treatments, growth rates were reduced by 60% in
low-pCO
2
cultures when compared with high-pCO
2
treatments. In support of these data,
gross:net N
2
-fixation rate ratios of C. watsonii (by acetylene reduction and
15
N
2
-isotope
uptake methods, respectively) reported by Garcia et al. (submitted) suggested that cellular
loss of fixed N increased as a function of decreasing pCO
2
and light (Mulholland et al.
2004, Mulholland 2007). In those experiments, net N
2
-fixation rates were highly
correlated with growth rates and implied that gross N
2
-fixation rates were in high excess
over the net N retained by cells when pCO
2
and light were low. Our finding here is
similar in that fixed N was likely in excess over that incorporated into cellular biomass in
the low pCO
2
, low light treatment. This interpretation is based on the small difference in
gross N
2
-fixation rates and the large difference in growth rates between pCO
2
treatments
at low light. Collectively, these data suggest that cellular growth rates can be decoupled
from CO
2
- and N
2
-fixation rates when cellular energy is low.
In P-limited cultures, P-uptake rates relative to growth rates were 3-12 times
higher in the low-light, low-pCO
2
treatment relative to other treatments. In the absence of
measurements of cellular P pools these high P-uptake:growth rate ratios are difficult to
interpret mechanistically, but may be related to a high cellular P demand for CO
2
and N
2
119
fixation when cellular energy is low. For instance, the cellular P content:N
2
fixation rate
ratio increased as a function of decreasing pCO
2
and light. This may be related to
differences in the turnover rate of ATP under various levels of cellular energy and
probably differences in relative ratios of cellular P pools such as rRNA, mRNA,
phospholipids and polyphosphates (polyP). For example, data from Orchard et al. (2011)
suggest that polyP:particulate C concentrations in Trichodesmium samples collected from
P-limited regions of the Sargasso Sea increased with increasing mixed layer depth and
associated decrease in mean mixed layer irradiance. Dyhrman and Haley (2006)
identified three genes in C. watsonii (WH8501) that are putatively involved with polyP
metabolism. PolyP ranges from 3 to 100’s of phosphate groups linked in chains by high-
energy ATP-like bonds and has a variety of known functions including energy storage
(Rao et al. 2009). Future studies might investigate diel shifts in cellular polyP
concentrations in C. watsonii, as polyP might be used to fuel dark metabolic processes
like N
2
fixation, particularly under slow growth conditions like low light and low pCO
2
.
Regardless of the relative proportions of these cellular P pools, our data suggest that the
total P quota can vary significantly based on relative changes in growth and P-uptake
rates.
Despite high cellular P quotas in low light treatments relative to high light
treatments, the P requirement for growth (K
1/2
) was 0.1 µM higher in high light
treatments when compared with low light treatments. This result seems counterintuitive
because a reduced P quota might be expected to reduce the P requirement for growth.
Although we do not understand the mechanisms that might have caused this shift, one
120
possibility comes from work by Berman-Frank et al. (2004) who documented high
oxidative stress and caspase activity (associated with programmed cell death) under high
light and low nutrient (Fe and P) conditions in Trichodesmium. Assuming that caspase
activity is induced at a low set-point concentration of P, additional caspase-inducing
stress, such as increased irradiance, could impact this set point. Thus, the T
cr
for viable
cell growth with respect to P may shift as a function of oxidative stress in C. watsonii.
We did not measure any parameters related to oxidative stress or programmed cell death
in our experiments but this is a direction for future research with C. watsonii as light and
P are major controlling factors of N
2
fixation in the oceans (Wu et al. 2000). Such an
interaction might result from low P quotas resulting from high growth rates under high
light. A low P quota might reduce the ability to quench photons. Another possibility is
that high irradiance may have an inhibitory effect on P acquisition in C. watsonii.
In summary, our data indicate that the effects of pCO
2
varied as a function of light
and P, and we surmise that these differences may be related to changes in the cellular
energy budget due to the CO
2
saturation states of CCMs. Our N
2
-fixation and growth rate
data at low light support findings by Garcia et al. (submitted) who documented positive
interactive effects of pCO
2
and light on cellular N retention by C. watsonii; cellular loss
of fixed N was correlated with slow growth and in general seems to be high when cellular
energy is low. In this regard, cellular retention of fixed N may be related to biochemical
changes that could affect the integrity of the cellular phospholipid membrane (Van Mooy
et al. 2006, 2009). This finding has implications for oceanic C flux, particularly in P-
limited waters where N
2
fixers dominate such as in the Atlantic Ocean and Mediterranean
121
Sea (Azam et al. 1983; Sañudo-Wilhelmy et al. 2001; Falcon et al. 2004; Mills et al.
2004; Sohm et al. 2008; Ridame et al. 2011).
In conclusion, these apparent effects of elevated pCO
2
on N
2
fixation by oceanic
unicellular diazotrophs may not be limited to C. watsonii but most likely do not apply to
all oceanic unicellular N
2
fixers. For instance, effects might be very different in non-
oxygenic oceanic diazotrophs (Falcon et al. 2004; Zehr et al. 2008), because these
organisms do not fix CO
2
and the primary benefit of high pCO
2
is directly associated
with CCMs. In this respect, areas where unicellular oxygenic diazotrophs can reach high
densities will likely be most affected by elevated pCO
2
, including tropical regions of the
Atlantic Ocean (Langlois et al. 2005; 2008) and parts of the Pacific Ocean near Hawaii
(Church et al. 2005; Goebel et al. 2007) and Northern Australia (Montoya et al. 2004).
In consideration of net effects of global change on unicellular oxygenic
diazotrophs, the expected decrease in P concentrations in stratified mixed layers of the
ocean will likely have negative effects on oceanic N
2
fixation (Hutchins et al. 2009).
These potential negative effects will likely be offset to some extent however, by the
concomitant increase in oceanic pCO
2
. With regard to previous research that
documented small effects of pCO
2
on N
2
-fixation rates by C. watsonii between present-
day and elevated 100-year predicted pCO
2
concentrations (Garcia et al. in press), we
expect the effect to be considerably stronger where P is low. The increase in average
light density of mixed layers is also expected to have positive effects on N
2
fixation with
increased retention of fixed N, but might result in a higher T
cr
for growth with respect to
P. In this respect, the combination of high light and low P concentrations may control
122
upper limits of growth and N
2
fixation by unicellular oxygenic diazotrophs, while lower
limits may be partially controlled by retention of fixed N.
Although recent experiments identified areas of P-limitation of N
2
fixation in the
North Pacific Subtropical Gyre (Watkins-Brandt et al. 2011), fixed P concentrations are
generally thought to be lower in the Atlantic relative to the Pacific Ocean (Wu et al.
2000; Deutsch et al. 2007), we expect the net effect of global change on N
2
fixation by
unicellular oxygenic diazotrophs to be more positive in the Pacific Ocean relative to that
in the Atlantic Ocean. This is primarily because higher P concentrations will allow
shallower upper limits of growth and N
2
fixation that will be forced by increased
stratification. Additionally, water column stratification is expected to increase as a
function of increasing temperature and freshwater input to the ocean surface. Thus, we
might expect stratification to be more intense in the Atlantic Ocean, further reducing P
concentrations in the upper layers of the ocean and intensifying negative pressures of
global change on N
2
fixation in the Atlantic Ocean relative to that in the Pacific. So far,
we anticipate both positive and negative effects of global change on growth and N
2
fixation by C. watsonii with regard to light, CO
2
and P concentrations. The extent of
these positive and negative effects are difficult to quantify and we must also consider
interactive effects of other key variables such as temperature and iron on oceanic N
2
-
fixation rates, as they might also share interdependent relationships with phosphorus,
CO
2
and light availability.
123
Chapter 3 References
Alley R.B., Berntsen, T., Bindoff, N.L., Chen, Z. & others. Summary for policymakers.
In: Solomon S, Qin D, Manning M, Chen, Z. and others. 2007. Climate change 2007: The
physical science basis. Contribution of Working Group I to the fourth assessment report
of the Intergovernmental Panel on Climate Change. Cambridge University Press,
Cambridge and New York.
Azam, F., Fenchel, T., Field, J.G., Gray, J.S., Meyer-Reil, L.A. & Thingstad, F. 1983.
The ecological role of water-column microbs in the sea. MEPS 10:257-267.
Badger, M. R., Price, G. D., Long, B. M. & Woodger, F. J. 2006. The environmental
plasticity and ecological genomics of the cyanobacterial CO
2
concentrating mechanism.
J. Exp. Bot. 57:249–265.
Barcelos e Ramos, J., Biswas, H., Schulz, K. G., LaRoche, J. & Riebesell, U. 2007.
Effect of rising atmospheric carbon dioxide on the marine nitrogen fixer Trichodesmium.
Glob. Biogeochem. Cycles 21:GB2028.
Berman-Frank, I., Quigg, A., Finkel, Z. V., Irwin, A. J. & Haramaty, L. 2007. Nitrogen-
fixation strategies and Fe requirements in cyanobacteria. Limnol. Oceanogr. 52:2260–
2269.
Breitbarth, E., Mills, M. M., Friedrichs, G. & LaRoche, J. 2004. The Bunsen gas
solubility coefficient of ethylene as a function of temperature and salinity and its
importance for nitrogen fixation assays. Limnol. Oceanogr.: Methods 2:282-288.
Chen, Y. B., Zehr, J. P. & Mellon, M. 1996. Growth and nitrogen fixation of the
diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp IMS 101
in defined media: Evidence for a circadian rhythm. J. Phycol. 32:916-923.
Church, M.J., Jenkins, B.D., Karl, D.M. & Zehr, J.P. 2005. Vertical distributions of
nitrogen–fixing phylotypes at Stn ALOHA in the oligotrophic North Pacific Ocean.
Aquat. Microb. Ecol. 38:3-14.
Deutsch, C., Sarmiento, J. L., Sigman, D. M., Gruber, N. & Dunne, J. P. 2007. Spatial
coupling of nitrogen inputs and losses in the ocean. Nature 445:163-167.
Donald, K.M., Scanlan, D.J., Carr, N.G., Mann, N.H. & Joint, I. 1997. Comparative
phosphorus nutrition of the marine cyanobacterium Synechococcus WH7803 and the
marine diatom Thalassiosira wessflogii. J. Planton Res. 19:1793-1813.
Dyhrman, S. T. & Haley, S.T. 2006. Phosphorus Scavenging in the Unicellular Marine
Diazotroph Crocosphaera watsonii. App. Environ. Microb. 72:1452-1458.
124
Falcon, L.I., Carpenter, E.J., Cipriano, F., Berman, B., & Capone, D.G. 2004. N
2
fixation
by unicellular bacterioplankton from the Atlantic and Pacific Oceans: Phylogeny and In
Situ Rates. Appl. Environ. Microbiol. 70:765-770.
Falcon, L.I., Pluvinage, S. & Carpenter, E.J. 2005. Growth kinetics of marine unicellular
N
2
-fixing cyanobacterial isolates in continuous culture in relation to phosphorus and
temperature. Mar. Ecol. Prog. Ser. 285:3-9.
Fu, F.-X., Zhang, Y., Bell, P.R.F. & Hutchins, D.A. 2005. Phosphate uptake and growth
kinetics of Trichodesmium (cyanobacteria) isolates from the North Atlantic Ocean and
the Great Barrier Reef, Australia. J. Phycol. 41:62-73.
Fu, F.-X., Mulholland, M. R., Garcia, N. S., Beck, A., Bernhardt, P. W., Warner, M. E.,
Sanudo-Wilhelmy, S. A. & Hutchins, D. A. 2008. Interactions between changing pCO
2
,
N
2
fixation, and Fe limitation in the marine unicellular cyanobacterium Crocosphaera.
Limnol. Oceanogr. 53:2472-2484.
Garcia, N.S., Fu, F-X., Breene, C.L., Yu, E.K., Bernhardt, P.W., Mulholland, M.R. &
Hutchins, D.A. (Submitted). Combined effects of CO
2
and irradiance on the unicellular
N
2
-fixing cyanobacterium Crocosphaera watsonii: A comparison of two isolates from the
western tropical Atlantic Ocean. Eur. J. Phycol.
Garcia, N. S., Fu, F-X, Breene, C. L., Bernhardt, P. W., Mulholland, M. R. Sohm, J. A. &
Hutchins, D. A. 2011. Interactive effects of irradiance and CO
2
on CO
2
- and N
2
fixation
in the diazotroph Trichodesmium erythraeum (Cyanobacteria). J. Phycol. 47:1292-1303.
Goebel, N.L., Edwards, C.A., Church, M.J. & Zehr, J.P. 2007. Modeled contributions of
three types of diazotrophs to nitrogen fixation at Station ALOHA. ISME J. 1:606-619.
Hutchins, D. A., Fu, F. X., Zhang, Y., Warner, M. E., Feng, Y., Portune, K., Bernhardt,
P. W. & Mulholland, M. R. 2007. CO
2
control of Trichodesmium N
2
-fixation,
photosynthesis, growth rates, and elemental ratios: Implications for past, present, and
future ocean biogeochemistry. Limnol. Oceanogr. 52:1293-1304.
Hutchins, D. A., Mulholland, M. R., & Fu, F.-X. 2009. Nutrient cycles and marine
microbes in a CO
2
enriched ocean. Oceanography 22:128-145.
Kranz, S. A., Levitan, O., Richter, K-U., Prášil, O., Berman-Frank, I., & Rost, B. 2010.
Combined effects of CO
2
and light on the N
2
-fixing cyanobacterium Trichodesmium
IMS101: Physiological responses. Plant Physiol. 154:334-345.
Kranz, S.A., Eichner, M. & Rost, B. 2011. Interactions between CCM and N
2
fixation in
Trichodesmium. Photosynth. Res. 109:73-84. DOI 10.1007/s11120-010-9611-3
125
Langlois, R.J., LaRoche, J., & Raab, P.A. 2005. Diazotrophic diversity and distribution in
the tropical and subtropical Atlantic Ocean. Appl. Environ. Microbiol. 71:7910. DOI:
10.1128/AEM.71.12.7910-7919.2005
Langlois, R.J., Hümmer, D. & LaRoche, J. 2008. Abundances and distributions of the
dominant nifH phylotypes in the northern Atlantic Ocean. Appl. Environ. Microbiol.
74:1922-1931. DOI: 10.1128/AEM.01720-07
Lebo, M.E., Sharp, J.H. 1992. Modeling phosphorus cycling in a well-mixed coastal plain
estuary. Estuar. Coastal Shelf Sci. 35:235-252.
Levitan, O., Rosenberg, G., Šetlík, I., Setlikova, E., Grigel, J., Klepetar, J., Prasil, O. &
Berman-Frank, I. 2007. Elevated CO
2
enhances nitrogen fixation and growth in the
marine cyanobacterium Trichodesmium. Glob. Change Biol. 13:531-538.
Mills, M. M., Ridame, C., Davey, M., La Roche, J. and Geider, R. J. 2004. Iron and
phosphorus co-limit nitrogen fixation in the eastern tropical North Atlantic. Nature
429:292-294.
Mohr, W., Intermaggio, M.P. & J. LaRoche. 2010. Diel rhythm of nitrogen and carbon
metabolism in the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii
WH8501. Environ. Microbiol. 12:412-421.
Moisander, P. H., Beinart, R. A., Hewson, I., White, A. E., Johnson, K. S., Carlson, C.
A., Montoya, J. P, & Zehr, J. P. 2010. Fixation domain unicellular cyanobacterial
distributions broaden the oceanic N
2
fixation domain. Science 327:1512-1514.
Montoya, J. P., Holl, C. M., Zehr J. P., Hansen, A., Villareal, T. A. & Capone, D. G.
2004. High rates of N
2
fixation by unicellular diazotrophs in the oligotrophic Pacific
Ocean. 430:1027-1031.
Morel, F. M. M., Rueter, J. G., Anderson, D. M. and Guillard, R. R. L. 1979. Aquil -
chemically defined phytoplankton culture-medium for trace-metal studies. J. Phycol.
15:135-141.
Mulholland, M. R., Bronk, D. A. & Capone, D. G. 2004. N
2
fixation and regeneration of
NH
4
+
and dissolved organic N by Trichodesmium IMS101. Aquatic Microb. Ecol. 37:85-
94.
Mulholland, M. R. 2007. The fate of nitrogen fixed by diazotrophs in the ocean.
Biogeosciences Discuss. 4:37-51.
126
Petit, J.R., Jouzel, J., Raynaud, D., Barkov, N.I., Barnola, J.-M., Basile, I., Bender, M.,
Chapellaz, J., Davis, M., Delaygue, G., Delmott, M., Kotlyakov, V.M., Legrand, M.,
Lipenkov, V.Y., Lorius, C., Pépin, L., Ritz, C., Saltzman, E. & Stievenard, M. 1999.
Climate and atmospheric history of the past 420,000 years from the Vostok ice core,
Antarctica. Nature 399:429-436.
Price, G. D., Badger, M. R., Woodger, F. J. & Long B. M. 2008. Advances in
understanding the cyanobacterial CO
2
-concentrating-mechanism (CCM): functional
components, Ci transporters, diversity, genetic regulation and prospects for engineering
into plants. J. Exp. Bot. 59:1441–1461.
Rao, N.N., Gomez-Garcia, M.R. & Kornberg, A. 2009. Inorganic polyphosphate:
essential for growth and survival. Annu. Rev. Biochem. 78:605-647.
Raven, J., Caldeira, K., Elderfield, H., Hoegh-Guldberg, O., Liss, P., Riebesell, U.,
Shepherd, H., Turley, C., & Watson, A. 2005. Ocean acidification due to increasing
atmospheric carbon dioxide. The Royal Society. Clyveden Press Ltd. Cardiff, UK
Ridame, C., Le Moal, M., Guieu, C., Ternon, E., Biegala, I.C., L’Helguen., S. & Pujo-
Pay, M. 2011. Nutrient control of N
2
fixation in the oligotrophic Mediterranean Sea and
the impact of Saharan dust events. Biogeosci. 8:2773-2783.
Saito, M.A., Bertrand, E.M., Dutkiewics, S., Bulygen, V.V., Moran, D.M., Montiero,
F.M., Follows, M.J., Valois, F.W. & J.B. Waterbury. 2011. Iron conservation by
reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii.
Proc. Natl. Acad. Sci. 108:2184-2189.
Sañudo-Wilhelmy, S. A., Kustka, A. B., Gobler, C. J., Hutchins, D. A., Yang, M., Lwiza,
K., Burns, J., Capone, D. G., Raven, J. A. & Carpenter, E. J. 2001. Phosphorus limitation
of nitrogen fixation by Trichodesmium in the central Atlantic Ocean. Nature 411: 66-69.
Scanlan, D.J., Mann, N.H. & Carr, N.G. 1993. The response of the picoplanktonic marine
cyanobacterium Synechococcus sp. WH7803 to phosphate starvation involves a protein
homologous to the periplasmic phosphate-binding protein of Escherichia coli. Mol.
Microbial. 10:181-191
Sohm, J. A., Mahaffey, C. & Capone, D. G. 2008. Assessment of relative phosphorus
limitation of Trichodesmium spp. in the North Pacific, North Atlantic, and the north coast
of Australia. Limnol. Oceanogr. 53:2495-2502.
Sohm, J.A., Edwards, B.R., Wilson, B.G., and E. A. Webb. 2011. Constitutive
extracellular polysaccharide (EPS) production by specific isolates of Crocosphaera
watsonii. Frontiers in Microbiology. 229. doi: 10.3389/fmicb.2011.00229
127
Van Mooy, B.A.S., Rocap, G. Fredericks, H.F., Evans, C.T., Devol, A.H. 2006.
Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in
oligotrophic marine environments. PNAS. 103: 8607-8612.
Van Mooy, B.S., Fredericks, H.F., Pedler, B.E. Dyhrman, S.T., Karl, D.M., Koblížek, M.,
Lomas, M.W., Mincer, T.J., Moore, L.R., Moutin, T., Rappé, M.S., Webb, E.A. 2009.
Phytoplankton in the ocean use non-phospholipids in response to phosphorus scarcity.
Nature. 458: 69-72.
Watkins-Brandt, K.S., Letelier, R.M., Spitz, Y.H., Church, M.J., Bottjer, D. & White,
A.E. 2011. Addition of inorganic or organic phosphorus enhances nitrogen and carbon
fixation in the oligotrophic North Pacific. Mar. Ecol. Prog. Ser. 432:17-29.
Wu, J. F., Sunda, W., Boyle, E. A. & Karl, D. M. 2000. Phosphate depletion in the
western North Atlantic Ocean. Science 289:759-762.
Zehr, J. P., Waterbury, J. B, Turner, P. J., Montoya, J. P., Omoregie, E., Steward, G. F.,
Hansen, A., & Karl, D. M. 2001. Unicellular cyanobacteria fix N
2
in the subtropical
North Pacific Ocean. Nature 412: 635-638.
Zehr, J.P., Bench, S.R., Carter, B.J., Hewson, I., Niazi, F., Shi, T., Tripp, J. & Affourtit,
J.P. 2008. Globally distributed uncultivated oceanic N
2
-fixing cyanobacteria lack
oxygenic photosystem II. Science 322:1110-1112.
128
Chapter 3 Table 1.
129
Chapter 3 Table 2.
130
Chapter 3 Table 3.
131
Chapter 3 Figures
Chapter 3 Figure 1. Saturation curve parameters were best fit to Crocosphaera watsonii
WH0003 irradiance response curves of specific growth rates (a), cellular CO
2
-fixation
rates (b), cellular N
2
-fixation rates (c), and cell diameter (d). *Data were removed from
calculations of Monod functional parameters.
Chapter 3 Figure 1.
132
Chapter 3 Figure 2. Specific growth rates (d
-1
) of Crocosphaera watsonii (WH0003) as a
function of phosphate concentration (0.1- 4 µM PO
4
3-
) in 190 ppm (triangles) and 800
ppm pCO
2
(circles) treatments under a) high (150 µmol quanta m
-2
s
-1
) and b) low (40
µmol quanta m
-2
s
-1
) irradiance. Functional response curves were best fit to means of
three experimental replicates. The standard error is plotted on means.
Chapter 3 Figure 2.
133
Chapter 3 Figure 3. Cellular CO
2
-fixation rates (fmol C cell
-1
hr
-1
) of Crocosphaera
watsonii (WH0003) as a function of phosphate concentration (0.1- 4 µM PO
4
3-
) in 190
ppm (triangles) and 800 ppm CO
2
(circles)
treatments under a) high (150 µmol quanta m
-2
s
-1
) and b) low (40 µmol quanta m
-2
s
-1
) irradiance; and gross N
2
-fixation rates (fmol N
cell
-1
hr
-1
) in the same phosphate and CO
2
treatments under c) high and d) low irradiance.
Functional response curves were best fit to means of three experimental replicates. The
standard error is plotted on means.
Chapter 3 Figure 3.
134
Chapter 3 Figure 4. a) Phosphate-uptake rates as a function of PO
4
3-
concentration (0.1-4
µM), in high light (150 µmol quanta m
-2
s
-1
) and low light (40 µmol quanta m
-2
s
-1
)
treatments grown under high pCO
2
(800 ppm, circles) and low pCO
2
(190 ppm,
triangles). b) Phosphate uptake rates relative to growth rates as a function of PO
4
3-
concentration (0.1-4 µM) in response to the same treatment matrix of light and pCO
2
. c)
Mean cellular P content collectively averaged from all P treatments at a given light-pCO
2
treatment. The standard error is plotted on treatment means.
Chapter 3 Figure 4.
135
Chapter 3 Figure 5. Cellular P demand for CO
2
fixation (a) and N
2
fixation (b) (moles of
cellular P per moles of CO
2
or N
2
fixed cell
-1
hr
-1
) by C. watsonii WH0003 in P-replete
cultures (4.0 µM) as a function of high light (open bars) and low light (filled bars), high
pCO
2
(800 ppm) and low pCO
2
(190 ppm). The standard error is plotted on treatment
means.
Chapter 3 Figure 5.
136
Chapter 4
SUMMARY AND CONCLUSIONS
In this final brief chapter to the dissertation, I summarize the major findings of my
research and highlight their implications for physiological mechanisms in oxygenic
diazotrophs. I also address the biogeochemical implications of my work by discussing
some of the ways in which I think global change will impact oceanic dinitrogen (N
2
)
fixation in a more acidified future ocean. Finally, I make inferences about how elevated
partial pressure of carbon dioxide (pCO
2
) might affect phytoplankton in general, by
applying underlying concepts of cellular physiological mechanisms with specific
reference to carbon concentrating mechanisms, and discuss potential avenues for future
research examining ocean acidification effects on biogeochemical cycles.
In the first chapter, I demonstrated that the effect of elevated pCO
2
on N
2
-fixation
rates by Trichodesmium erythraeum is dependent on light intensity. This finding is
shared by another phytoplankton physiology research group in Germany who
documented very similar trends in the same strain of T. erythraeum (Kranz et al. 2011).
In addition to these two laboratory studies, some of our limited field data in the Gulf of
Mexico suggest that this trend might extend into the natural environment. Briefly, the
data suggest that N
2
-fixation rates by Trichodesmium will be highly impacted by elevated
pCO
2
in deeper layers of the water column with relatively small effects near the surface,
where photon flux is high. This finding suggests that field abundances of Trichodesmium
may be shifted to lower regions of the water column in the future ocean. This niche shift
137
could also potentially create new sub-habitats for other groups of bacterioplankton,
phytoplankton and zooplankton.
Recently, unpublished data from Hutchins et al. (manuscript in preparation)
indicated that effects of pCO
2
on N
2
fixation by Trichodesmium and Crocosphaera are
species- and strain specific. Future research, perhaps focusing on differences in carbon
acquisition mechanisms between strains, will need to corroborate this finding as there
may be genetic, geographical and biogeochemical patterns that are related to these
differences. If we can identify some of these patterns we might begin to make
connections and interpretations about how elevated pCO
2
will impact N
2
fixation by
diazotrophic cyanobacteria as a group. Along with this, however, we still need to
identify interactive effects of other environmental variables on oceanic N
2
fixation. For
example, interactive effects of iron (Fe) and phosphorus (P) availability on N
2
fixation by
oceanic N
2
fixers have not yet been well described. This interaction has received some
attention in field studies by Mills et al. (2004) and Ridame et al. (2011) but no studies
have investigated the specific type of relationship between these two major controlling
nutrients that limit N
2
-fixation rates in the surface layers of the world’s ocean (Wu et al.
2000).
In the second chapter, I identified differences in effects of light and interactive
effects of light and pCO
2
on growth, CO
2
- and N
2
fixation by large (WH0402) and small
(WH0401) strains of Crocosphaera watsonii isolated from the western tropical Atlantic
Ocean. This study documented large differences in maximum growth rates between
these strains and suggested that these strains may have different CO
2
requirements for
138
growth, CO
2
- and N
2
-fixation rates. For example, these rates were not limited by low
pCO
2
(190 ppm) in the strain with large cells but were in the strain with small cells.
Unpublished data by Hutchins et al. (manuscript in preparation) also identified
differences in N
2
-fixation rates between strains of C. watsonii, similar to the findings for
Trichodesmium.
In general, results of my experiments also suggest that N
2
fixation by these two
strains of C. watsonii were not limited by present-day atmospheric pCO
2
. This finding is
in contrast to the study by Fu et al. (2008) who documented a relatively large increase in
growth and N
2
-fixation rates of another strain (WH8501) in response to elevated 100-year
predicted pCO
2
when compared to the present-day pCO
2
. Part of the overall goal of this
second study was to compare the interactive effects of light and pCO
2
on N
2
fixation by
Crocosphaera with those on N
2
fixation by Trichodesmium. This comparison was
difficult to achieve, however, due to the low pCO
2
requirement of the large strain and the
inherent difficulty of maintaining viable cultures of the small strain. Thus, I attempted to
address light and pCO
2
interactions in the following study, along with interactive effects
with phosphorus (P). Collectively, these studies reiterate the need to examine species-
specific and strain-specific differences in carbon acquisition mechanisms in these two
biogeochemically relevant genera.
Another one of the major findings of this second-chapter study was that the
gross:net N
2
-fixation rate ratio (a proxy for cellular retention of fixed N) varied as a
function of light and pCO
2
in C. watsonii. Coincidentally, I also documented a change in
this ratio as a function of light in Trichodesmium. These data suggest that cellular
139
retention of fixed N declines when growth is limited by low energy conditions. One
mechanistic explanation for this might be related to a compromised integrity of the cell
membrane when energy is low.
In the third study, I examined interactive effects of P, light and pCO
2
on N
2
-
fixation rates by a strain of C. watsonii with large cells (WH0003). The main findings of
this study were primarily that all three of these environmental variables are dependent on
each other in their colimitation of growth, CO
2
- and N
2
-fixation rates by C. watsonii.
Specifically, elevated pCO
2
acted to decrease the half-saturation constant (K
1/2
) and
critical threshold (T
cr
) concentration for growth with respect to P, while elevated light
acted to increase the P concentration for K
1/2
and T
cr
. In addition, I was finally able to
clarify the interactive effect of light and pCO
2
on N
2
fixation by Crocosphaera that I had
tried to identify with only partial success in my second study.
The ways in which all three of these dependent relationships interact to affect
these rate parameters were not expected. For instance, although the effects of light and
pCO
2
on N
2
-fixation rates by T. erythraeum were dependent on each other, the effect in
Crocosphaera was reversed; the effect of pCO
2
on N
2
fixation was highest under low
light in T. erythraeum and highest under high light in Crocosphaera. Secondly, Hutchins
et al. (2007) documented nearly identical percent increases in N
2
-fixation rates as a
function of elevated pCO
2
in both P-replete and P-limited cultures of T. erythraeum,
indicating that P and pCO
2
affected N
2
-fixation rates independently of each other. This
contrasts strongly with my results showing an interdependence of P and CO
2
availability
in Crocosphaera. Lastly, I observed a high T
cr
for growth, CO
2
- and N
2
fixation with
140
respect to P concentration under high light in comparison with low light. This shift is
counterintuitive, considering the low cellular P quota:N
2
fixation rate ratio in high light
cultures in comparison with low light cultures. Although the interactive effects of P and
light on Trichodesmium have not been examined, the results from this experiment leave
little reason to assume that they would be similar to the effects on Crocosphaera. The
reason for the differential interactive effects of light, pCO
2
and P between these species is
not known but might be related to the different N
2
-fixation strategies that they use (i.e.
Trichodesmium fixes N
2
and CO
2
simultaneously during the photoperiod while
Crocosphaera separates these processes temporally).
Alternatively, the interaction of P and CO
2
might elicit different effects on N
2
fixation under variable light conditions. For example, the major conclusion brought forth
by Kranz et al. (2010) and Garcia et al. (2011) suggested that cellular energy shifts
between CCMs and N
2
fixation processes as a function of light and pCO
2
. Thus, the
interactive effects of pCO
2
and P may mimic an independent relationship at a mid level
irradiance (where the Hutchins study was conducted) but might actually appear as a
dependent relationship under variable light conditions.
Results from this third study also suggest that even though the effect of pCO
2
elevated from present-day to 100 year predicted levels did not elicit a strong stimulation
of N
2
fixation rates in two strains of C. watsonii, the general effect of elevated pCO
2
was
strongest under low P conditions. This has important implications for the actual effect of
CO
2
on N
2
-fixation rates in the field, because typically concentrations of phosphorus are
relatively low. Future projected stratification increases are likely to lower available P in
141
the euphotic zone even further. Thus, it is difficult to predict the magnitude of the effect
of elevated pCO
2
on Crocosphaera. Perhaps the most meaningful information would
come from direct studies of elevated pCO
2
on Crocosphaera in the field. This approach,
however, is further complicated by the fact that some strains of unicellular oxygenic
diazotrophs are perhaps non-responsive to increases in pCO
2
above present-day
concentrations, at specific concentrations of P. One way to investigate this might be to
artificially reduce the P concentration in field manipulation experiments by concentrating
cells in a diluted medium.
In general, perhaps the strongest impact of global change on N
2
-fixation rates by
Crocosphaera will be the effect of brighter light in shallower, more stratified mixed
layers. My studies suggest that at present, the upper limits of a Crocosphaera bloom
(near 50 meters depth according to Goebel et al. 2007, equivalent to around 200 µmol
quanta m
-2
s
-1
according to data by Breitbarth et al. 2008) might be set by a diminishing
concentration of P with proximity to the surface and increasing light. This suggestion is
based on the high T
cr
for growth with respect to P under high light, relative to low light.
Thus, as the water column becomes more stratified due to warming and increased
freshwater input to the oceans, Crocosphaera might be able to cope with stronger light
concentrations due to the concomitant rise in pCO
2
and the associated decrease in T
cr
for
growth with respect to P. While my data suggest that this might happen, the
concentration of P is expected to decrease with increasing stratification, thereby possibly
offsetting this positive effect of elevated pCO
2
in enabling Crocosphaera to fix N
2
under
increased light density.
142
Thus, these three studies have elucidated some interactive effects between light
pCO
2
and P in a variety of strains and species of oceanic oxygenic diazotrophs.
Collectively, these interactive effects identified both positive and negative potential
impacts of global change on oceanic N
2
fixation. Studies by Goebel et al. (2007) and
Langlois et al. (2005, 2008) suggest that unicellular oxygenic diazotrophs probably
represent near ~10% of the total N
2
fixation in regions of the tropical Pacific and Atlantic
Oceans while Trichodesmium most likely contributes substantially more, near ~50%. In
addition, Moisander et al. (2010) indicated that the range of non-oxygenic unicellular
diazotrophs is probably much larger than originally thought, with range estimates
expanding into deeper water and higher latitudes. Thus, the field must focus on studies
of non-oxygenic unicellular diazotrophs, as little is known about the potential effects of
global change on these organisms due to the lack of available cultures.
Finally, future studies must also focus on the effects of iron. So far, we know
little about how iron will interact with other environmental factors that are likely to
change in the future. For instance, while the interactive effect of elevated pCO
2
and Fe
have been studied in Crocosphaera by Fu et al. (2008), no study has investigated this
interaction in Trichodesmium. We also need examine interactive effects of Fe and P on
N
2
fixation in oceanic diazotrophs.
In conclusion, these studies have brought the field closer to making accurate
predictions with regard to positive and negative effects of global change on oceanic N
2
fixation. Because the N cycle is tightly linked to the C cycle through the biology of the
ocean, a clear understanding of the N cycle is necessary to predict impacts of global
143
change on the C cycle. In an effort to understand biological responses to global change,
some of the conclusions of these studies may apply to non-N
2
-fixing clades of
phytoplankton, particularly those with carbon concentrating mechanisms. Thus the
design of these studies of interactive effects of multiple variables might be applied to a
broad range of phytoplankton in an effort to model effects of global change on
biogeochemical cycles in the future changing ocean.
144
Comprehensive List of References
Alley RB, Berntsen T, Bindoff NL, Chen Z and others. Summary for policymakers. In:
Solomon S, Qin D, Manning M, Chen Z and others (Eds.) Climate change 2007: The
physical science basis. Contribution of Working Group I to the fourth assessment report
of the Intergovernmental Panel on Climate Change. Cambridge University Press,
Cambridge and New York. (2007).
Azam, F., Fenchel, T., Field, J.G., Gray, J.S., Meyer-Reil, L.A. & Thingstad, F. 1983.
The ecological role of water-column microbs in the sea. MEPS 10:257-267.
Badger, M. R., Andrews, T. J., Whitney, S. M., Ludwig, M., Yellowlees, D. C., Leggat,
W. & Price, G. D. 1998. The diversity and coevolution of RubisCO, plastids, pyrenoids,
and chloroplast based CO
2
-concentrating mechanisms in algae. Can. J. Bot. 76:1052-
1071.
Badger, M. R., Price, G. D., Long, B. M. & Woodger, F. J. 2006. The environmental
plasticity and ecological genomics of the cyanobacterial CO
2
concentrating mechanism.
J. Exp. Bot. 57:249–265.
Barcelos e Ramos, J., Biswas, H., Schulz, K. G., LaRoche, J. & Riebesell, U. 2007.
Effect of rising atmospheric carbon dioxide on the marine nitrogen fixer Trichodesmium.
Glob. Biogeochem. Cycles 21:GB2028.
Bates, N. R. 2001. Interannual variability of oceanic CO
2
and biogeochemical properties
in the Western North Atlantic subtropical gyre. Deep-Sea Res. II 48:1507-1528.
Bell, P. R. F. & Fu, F-X. 2003. Effect of light on growth, pigmentation and N
2
fixation of
cultured Trichodesmium sp. from the Great Barrier Reef lagoon. Hydrobiologia 543:25–
35.
Behrenfeld, M. J., O’Malley, R. T., Siegel, D. A., McClain, C. R., Sarmiento, J. L.,
Feldman, G. C., Milligan, A. J., Falkowski, P. G., Letelier, R. M., & Boss, E. S. 2006.
Climate-driven trends in contemporary ocean productivity. Nature 444:752-755.
Beman, J. M., Chow, C.-E., King, A. L., Feng, Y., Fuhrman, J. A., Anderson, A. Bates,
N. R., Popp, B. N. & Hutchins, D. A. 2010. Global declines in oceanic nitrification rates
as a consequence of ocean acidification. PNAS, USA. 108:208-213.
Berman-Frank, I., Lundgren, P., Chen, Y-B., Kupper, H., Kolber, Z., Bergman, B. &
Falkowski, P. 2001. Segregation of nitrogen fixation and oxygenic photosynthesis in the
marine cyanobacterium Trichodesmium. Science 294:1534-1537
145
Berman-Frank, I., Quigg, A., Finkel, Z. V., Irwin, A. J. & Haramaty, L. 2007. Nitrogen-
fixation strategies and Fe requirements in cyanobacteria. Limnol. Oceanogr. 52:2260–
2269
Boyd, P. W. & Doney, S. C. 2002. Modeling regional responses by marine pelagic
ecosystems to global climate change. Geophys. Res. Lett. 29:1806.
Boyd, P. W., Strzepek, R., Fu, F.-X. & Hutchins, D.A. 2010. Environmental control of
open ocean phytoplankton groups: now and in the future. Limnol. Oceanogr.
55: 1353–
1376.
Breitbarth, E., Mills, M. M., Friedrichs, G. & LaRoche, J. 2004. The Bunsen gas
solubility coefficient of ethylene as a function of temperature and salinity and its
importance for nitrogen fixation assays. Limnol. Oceanogr.: Methods 2:282-288.
Breitbarth, E., Oschlies, A., & LaRoche J. 2007. Physiological constraints on the global
distribution of Trichodesmium - effect of temperature on diazotrophy. Biogeosciences.
4:53-61.
Breitbarth, E., Wohlers, J., Klas, J., LaRoche, J. & Peeken, I. 2008. Nitrogen fixation
and growth rates of Trichodesmium IMS-101 as a function of light intensity. Mar. Ecol.
Prog. Ser. 359:25-36.
Caldeira, K. & Wickett, M.E. 2003. Anthropogenic carbon and ocean pH. Nature.
425:365.
Capone, D. G. 1993. Determination of nitrogenase activity in aquatic samples using the
acetylene reduction procedure. In: Kemp, P. F., J. J. Cole, B. F. Sherr, E. B. Sherr (eds).
Handbook of methods in aquatic microbial ecology. Lewis Publishers, Boca Raton, FL, p
621–631.
Capone, D. G., Ferrier, M. D., & Carpenter, E. J. 1994. Amino acid cycling in colonies of
the planktonic marine cyanobacterium Trichodesmium thiebautii. Appl. Env. Microbiol.
60:3989-3995.
Capone, D. G., Zehr, J. P., Paerl, H. W., Bergman, B. & Carpenter, E. J. 1997.
Trichodesmium, a globally significant marine cyanobacterium. Science 276:1221-1229.
Capone, D. G., Burns, J. A., Montoya, J. P., Subramaniam, A., Mahaffey, C., Gunderson,
T., Michaels, A. F. & Carpenter, E. J. 2005. Nitrogen fixation by Trichodesmium spp.:
An important source of nitrogen to the tropical and subtropical North Atlantic Ocean.
Global Biogeochem. Cy. 19:GB2024.
Capone, D. G. 2008. The marine nitrogen cycle. Microbe 3:186-192.
146
Carpenter, E. J. & C. C. Price. 1977. Nitrogen fixation , distribution and production of
Oscillatoria (Trichodesmium) spp. in the western Sargasso and Caribbean Seas. Limnol.
Oceanogr. 22:60-72.
Carpenter, E. J., O’Neil, J. M., Dawson, R., Capone, D. G., Siddiqui, P. J. A.,
Roenneberg, G. T. & Bergman, B. 1993. The tropical diazotrophic phytoplanktonkter
Trichodesmium: biological characteristics of two common species. Mar. Ecol. Prog. Ser.
95:295-304.
Chen, Y. B., Zehr, J. P. & Mellon, M. 1996. Growth and nitrogen fixation of the
diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp IMS 101
in defined media: Evidence for a circadian rhythm. J. Phycol. 32:916-923.
Church, M.J., Jenkins, B.D., Karl, D.M. & Zehr, J.P. 2005. Vertical distributions of
nitrogen–fixing phylotypes at Stn ALOHA in the oligotrophic North Pacific Ocean.
Aquat. Microb. Ecol. 38:3-14.
Church, M. J., Björkman, K. M., Karl, D. M., Saito, M. A. & Zehr, J. P. 2008. Regional
distributions of nitrogen-fixing bacteria in the Pacific Ocean. 53:63-77.
Davis, C. S. & McGillicuddy, D. J. 2006. Transatlantic abundance of the N
2
-fixing
colonial cyanobacterium Trichodesmium. Science 312:1517-1519.
Dickson, A. G., & Millero, F. J. 1987. A comparison of the equilibrium constants for the
dissociation of carbonic acid in seawater media. Deep-Sea Res. 34:1733-1743.
Deutsch, C., Sarmiento, J. L., Sigman, D. M., Gruber, N. & Dunne, J. P. 2007. Spatial
coupling of nitrogen inputs and losses in the ocean. Nature 445:163-167.
Donald, K.M., Scanlan, D.J., Carr, N.G., Mann, N.H. & Joint, I. 1997. Comparative
phosphorus nutrition of the marine cyanobacterium Synechococcus WH7803 and the
marine diatom Thalassiosira wessflogii. J. Planton Res. 19:1793-1813.
Dyhrman, S. T. & Haley, S.T. 2006. Phosphorus Scavenging in the Unicellular Marine
Diazotroph Crocosphaera watsonii. App. Environ. Microb. 72:1452-1458.
Dyhrman S. T., Chappell, P. D., Haley, S. T., Moffett, J. W., Orchard, E. D., Waterbury,
J. B. & Webb, E. A. 2006. Phosphonate utilization by the globally important marine
diazotroph Trichodesmium. Nature 439:1452-1458.
Falcon, L.I., Carpenter, E.J., Cipriano, F., Berman, B., & Capone, D.G. 2004. N
2
fixation
by unicellular bacterioplankton from the Atlantic and Pacific Oceans: Phylogeny and In
Situ Rates. Appl. Environ. Microbiol. 70:765-770.
147
Falcon, L.I., Pluvinage, S. & Carpenter, E.J. 2005. Growth kinetics of marine unicellular
N
2
-fixing cyanobacterial isolates in continuous culture in relation to phosphorus and
temperature. Mar. Ecol. Prog. Ser. 285:3-9.
Falkowski, P. G. 1997. Evolution of the nitrogen cycle and its influence on the biological
sequestration of CO
2
in the ocean. Nature 387:272-275.
Falkowski, P.G., Barber, R.T. & Smetacek, V. 1998. Biogeochemical controls and
feedbacks on ocean primary production. Science. 281:200-206.
Fu, F.-X. & Bell, P. R. F. 2003. Growth N
2
-fixation and photosynthesis in a
cyanobacterium, Trichodesmium sp., under Fe stress. Biotech. Lett. 25, 645-649.
Fu, F.-X., Zhang, Y., Bell, P. R. F. & Hutchins, D. A. 2005. Phosphate uptake and
growth kinetics of Trichodesmium (Cyanobacteria) isolates from the North Atlantic
Ocean and the Great Barrier Reef, Australia. J. Phycol. 41:62-73.
Fu, F-X., Warner, M. E., Zhang, Y., Feng, Y. & Hutchins, D. A. 2007. Effects of
increased temperature and CO
2
on photosynthesis, growth and elemental ratios in marine
Synechococcus and Prochlorococcus (Cyanobacteria). J. Phycol. 43:485-496.
Fu, F.-X., Mulholland, M. R., Garcia, N. S., Beck, A., Bernhardt, P. W., Warner, M. E.,
Sanudo-Wilhelmy, S. A. & Hutchins, D. A. 2008. Interactions between changing pCO
2
,
N
2
fixation, and Fe limitation in the marine unicellular cyanobacterium Crocosphaera.
Limnol. Oceanogr. 53:2472-2484.
Garcia, N. S., Fu, F-X, Breene, C. L., Bernhardt, P. W., Mulholland, M. R. Sohm, J. A. &
Hutchins, D. A. 2011. Interactive effects of irradiance and CO
2
on CO
2
- and N
2
fixation
in the diazotroph Trichodesmium erythraeum (Cyanobacteria). J. Phycol. 47:1292-1303.
Garcia, N.S., Fu. F., Yu, E.K., Breene, C.L., Bernhardt, P.W., Mulholland, M.R.,
Hutchins, D.A. 2013. Combined effects of CO
2
and light on the unicellular N
2
-fixing
cyanobacterium Crocosphaera watsonii: A comparison of two isolates from the western
tropical Atlantic Ocean. Eur. J. Phycol. In press.
Gallon, J. R. 1981. The oxygen sensitivity of nitrogenase: a problem for biochemists and
micro-organisms. Trends Biochem. Sci., 6, 19-23.
Goebel, N.L., Edwards, C.A., Church, M.J. & Zehr, J.P. 2007. Modeled contributions of
three types of diazotrophs to nitrogen fixation at Station ALOHA. ISME J. 1:606-619.
148
Goebel, N. L., Edwards, C. A., Carter, B. J., Achilles, K. M., & Zehr, J. P. 2008. Growth
and carbon content of three different-sized diazotrophic cyanobacteria observed in the
subtropical North Pacific. J. Phycol. 44:1212-1220.
Howard, J.B. & Reese, D.C. 1996. Structural basis of biological nitrogen fixation. Chem.
Rev. 96:2965-2982.
Hutchins, D. A., Fu, F. X., Zhang, Y., Warner, M. E., Feng, Y., Portune, K., Bernhardt,
P. W. & Mulholland, M. R. 2007. CO
2
control of Trichodesmium N
2
-fixation,
photosynthesis, growth rates, and elemental ratios: Implications for past, present, and
future ocean biogeochemistry. Limnol. Oceanogr. 52:1293-1304.
Hutchins, D. A. & Fu, F.-X. 2008. Linking the oceanic biogeochemistry of iron and
phosphorus with the marine nitrogen cycle. pp. 1627-1653. In: Nitrogen in the Marine
Environment, 2
nd
edition. D. G. Capone, D. A. Bronk, M. R. Mulholland and E. J.
Carpenter [Eds.], Elsevier Press, Amsterdam.
Hutchins, D. A., Mulholland, M. R., & Fu, F.-X. 2009. Nutrient cycles and marine
microbes in a CO
2
enriched ocean. Oceanography 22:128-145.
Intergovernmental Panel on Climate Change (IPCC) – Working Group 1 (2007). Climate
Change 2007: The Physical Science Basis. Cambridge University Press.
Karl, D., Michaels, A., Bergman, B., Capone, D., Carpenter, E., Letelier, R., Lipschultz,
F., Paerl, H., Sigman, D. & Stal, L. 2002. Dinitrogen fixation in the world’s oceans.
Biogeochem. 57/58:47-98.
Karl, D., Letelier, R., Tupas, L., Dore, J., Christian, J. & Hebel, D. 1997. The role of
nitrogen fixation in biogeochemical cycling in the subtropical North Pacific Ocean.
Nature 388:533-538.
Karl, D.M., Letelier, R., Hebel, D., Tupas, L., Dore, J., Christian, J. & Winn, C. 1995.
Ecosystem changes in the North Pacific subtropical gyre attributed to the 1991-92 El
Niño. Nature. 373:230-234.
King, A. L., Sanudo-Wilhelmy, S. A., Leblanc, K., Hutchins, D. A. & Fu, F.-X. 2011.
CO
2
and vitamin B
12
interactions determine bioactive trace metal requirements of a
subarctic Pacific diatom. ISME 5:1388-1396. doi:10.1038/ismej.2010.211.
Kranz, S. A., Sultemeyer, D., Richter, K. U., & Rost, B. 2009. Carbon acquisition by
Trichodesmium: The effect of pCO
2
and diurnal changes. Limnol. Oceanogr. 54:548-559.
149
Kranz, S. A., Levitan, O., Richter, K-U., Prášil, O., Berman-Frank, I., Rost, B. 2010.
Combined effects of CO
2
and light on the N
2
-fixing cyanobacterium Trichodesmium
IMS101: Physiological responses. Plant Physiol. 154:334-345.
Kranz, S.A., Eichner, M. & Rost, B. 2011. Interactions between CCM and N
2
fixation in
Trichodesmium. Photosynth. Res. 109:73-84. DOI 10.1007/s11120-010-9611-3
Kustka, A. B., Sanudo-Wilhelmy, S. A., Carpenter, E. J., Capone, D., Burns, J. & Sunda,
W. G. 2003. Iron requirements for dinitrogen- and ammonium-supported growth in
cultures of Trichodesmium (IMS 101): Comparison with nitrogen fixation rates and iron:
carbon ratios of field populations. Limnol. Oceanogr. 48:1869-1884.
Langlois, R.J., LaRoche, J., & Raab, P.A. 2005. Diazotrophic diversity and distribution in
the tropical and subtropical Atlantic Ocean. Appl. Environ. Microbiol. 71:7910. DOI:
10.1128/AEM.71.12.7910-7919.2005
Langlois, R.J., Hümmer, D. & LaRoche, J. 2008. Abundances and distributions of the
dominant nifH phylotypes in the northern Atlantic Ocean. Appl. Environ. Microbiol.
74:1922-1931. DOI: 10.1128/AEM.01720-07
Leblanc, K., Hare, C.E., Boyd, P.W., Bruland, K.W., Sohst, B., Pickmere, S., Lohan,
M.C., Buck, K., Ellwood, M. & Hutchins, D.A. 2005. Deep Sea Res. I. 52:1842-1864.
Lebo, M.E., Sharp, J.H. 1992. Modeling phosphorus cycling in a well-mixed coastal plain
estuary. Estuar. Coastal Shelf Sci. 35:235-252.
Levitan, O., Rosenberg, G., Šetlík, I., Setlikova, E., Grigel, J., Klepetar, J., Prasil, O. &
Berman-Frank, I. 2007. Elevated CO
2
enhances nitrogen fixation and growth in the
marine cyanobacterium Trichodesmium. Glob. Change Biol. 13:531-538.
Levitan, O., Kranz, S. A., Spungin, D., Prášil, O., Rost, B. & Berman-Frank, I. 2010.
Combined effects of CO
2
and light on the N
2
-fixing cyanobacterium Trichodesmium
IMS101: A mechanistic view. Plant Physiol. 154:346-356.
Lewis, E. and D. W. R. Wallace 1998. Program Developed for CO
2
System Calculations.
ORNL/CDIAC-105. Carbon Dioxide Information Analysis Center, Oak Ridge National
Laboratory, U.S. Department of Energy, Oak Ridge, Tenessee. Available at:
http://cdiac.ornl.gov/oceans/co2rprt.html (last accessed 20 March 2011).
Mague, T. H., Mague, C., & Holm-Hansen, O. 1977. Physiology and chemical
composition of nitrogen-fixing phytoplankton in the central North Pacific Ocean. Mar.
Biol. 24:109-119.
150
Mahowald, N.M., Yoshioka, M., Collins, W.D., Conley, A.J., Fillmore, D.W. &
Coleman, D.B. 2006. Climate response and radiative forcing from mineral aerosols
during the last glacial maximum, pre-industrial, current and doubled-carbon dioxide
climates. Geophys. Res. Lett. 33:L20705. DOI: 10.1029/2006GL026126.
Mehrbach, Y., Culberson, C., Hawley, J. & Pytkovicz, R. 1973. Measurement of the
apparent dissociation constants of carbonic acid in seawater at atmospheric pressure.
Limnol. Oceanogr. 18:897-907.
Michaels, A. F., Karl, D. M. & Capone, D. G. 2001. Element stoichiometry, new
production and nitrogen fixation. Oceanography 14:68–77.
Mills, M. M., Ridame, C., Davey, M., La Roche, J. and Geider, R. J. 2004. Iron and
phosphorus co-limit nitrogen fixation in the eastern tropical North Atlantic. Nature
429:292-294.
Moisander, P. H., Beinart, R. A., Hewson, I., White, A. E., Johnson, K. S., Carlson, C.
A., Montoya, J. P, & Zehr, J. P. 2010. Unicellular cyanobacterial distributions broaden
the oceanic N
2
fixation domain. Science 327:1512-1514.
Montoya, J. P., Voss, M., Kähler, P. & Capone, D. G. 1996. A simple, high-precision,
high-sensitivity tracer assay for N
2
fixation. Appl. and Environ. Microbiol. 62:986-993.
Montoya, J. P., Holl, C. M., Zehr J. P., Hansen, A., Villareal, T. A. & Capone, D. G.
2004. High rates of N
2
fixation by unicellular diazotrophs in the oligotrophic Pacific
Ocean. 430:1027-1031.
Moore, C. M., Mills, M.M., Achterberg, E.P.,Geider, R.J., LaRoche, J., Lucas, M.I.,
McDonagh, E.L., Pan, X., Poulton, A.J., Rijkenberg, M. J. A., Suggett, D.J., Ussher, S.J.,
Woodward, E.M.S. 2009. Large-scale distribution of Atlantic nitrogen fixation
controlled by iron availability. Nature Geoscience 12: 867-871.
Morel, F. M. M., Rueter, J. G., Anderson, D. M. and Guillard, R. R. L. 1979. Aquil -
chemically defined phytoplankton culture-medium for trace-metal studies. J. Phycol.
15:135-141.
Mohr, W., Intermaggio, M.P. & J. LaRoche. 2010a. Diel rhythm of nitrogen and carbon
metabolism in the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii
WH8501. Environ. Microbiol. 12:412-421.
Mohr, W., Großkopf, T., Wallace, D.W.R. & J. LaRoche. 2010b. Methodological
underestimation of oceanic nitrogen fixation rates. PLoS ONE. 5:e12583.
Monod, J. 1949. The growth of bacterial cultures. Annu. Rev. Microbiol. 3:371-394.
151
Mulholland, M. R. 2007. The fate of nitrogen fixed by diazotrophs in the ocean.
Biogeosciences Discuss. 4:37-51.
Mulholland, M. R. and Bernhardt, P. W. 2005. The effect of growth rate, phosphorus
concentration and temperature on N
2
-fixation, carbon fixation, and nitrogen release in
continuous cultures of Trichodesmium IMS101. Limnol. Oceangr. 50:839-849.
Mulholland, M. R., Bronk, D. A. & Capone, D. G. 2004. N
2
fixation and regeneration of
NH
4
+
and dissolved organic N by Trichodesmium IMS101. Aquatic Microb. Ecol. 37:85-
94.
Mulholland, M.R., Bernhardt, P.W. Blanco-Garcia, J.L., Manino, A., Hyde, K.,
Mondragon, E. Turk, K., Moisander, P.H. & Zehr, J.P. 2012. Rates of dinitrogen fixation
and the abundance of diazotrophs in North American coastal waters between Cape
Hatteras and Georges Bank. Limnol. Oceanogr. (in press).
Petit, J.R., Jouzel, J., Raynaud, D., Barkov, N.I., Barnola, J.-M., Basile, I., Bender, M.,
Chapellaz, J., Davis, M., Delaygue, G., Delmott, M., Kotlyakov, V.M., Legrand, M.,
Lipenkov, V.Y., Lorius, C., Pépin, L., Ritz, C., Saltzman, E. & Stievenard, M. 1999.
Climate and atmospheric history of the past 420,000 years from the Vostok ice core,
Antarctica. Nature 399:429-436.
Price, G. D., Badger, M. R., Woodger, F. J. & Long B. M. 2008. Advances in
understanding the cyanobacterial CO
2
-concentrating-mechanism (CCM): functional
components, Ci transporters, diversity, genetic regulation and prospects for engineering
into plants. J. Exp. Bot. 59:1441–1461.
Prufert-Bebout, L., Paerl, H. W., & Lassen, C. 1993. Growth, nitrogen fixation, and
spectral attenuation in cultivated Trichodesmium species. Appl. Env. Microb. 59:1367-
1375.
Rao, N.N., Gomez-Garcia, M.R. & Kornberg, A. 2009. Inorganic polyphosphate:
essential for growth and survival. Annu. Rev. Biochem. 78:605-647.
Raven, J., Caldeira, K., Elderfield, H., Hoegh-Guldberg, O., Liss, P., Riebesell, U.,
Shepherd, H., Turley, C., & Watson, A. 2005. Ocean acidification due to increasing
atmospheric carbon dioxide. The Royal Society. Clyveden Press Ltd. Cardiff, UK
Redfield, A. C., B. H. Ketchum, and F. A. Richards, The influence of organisms on the
composition of sea-water, in The Sea, edited by M. N. Hill, vol. 2, pp. 26–77, Wiley-
Interscience, New York, 1963.
152
Ridame, C., Le Moal, M., Guieu, C., Ternon, E., Biegala, I.C., L’Helguen., S. & Pujo-
Pay, M. 2011. Nutrient control of N
2
fixation in the oligotrophic Mediterranean Sea and
the impact of Saharan dust events. Biogeosci. 8:2773-2783.
Riebesell, U., Fabry, V. J., Hansson, L., Gattuso, J. P. 2010. Guide to best practices for
ocean acidification research and data reporting. Publications Office of the European
Union, Luxembourg, 258 pp.
Saito, M.A., Bertrand, E.M., Dutkiewics, S., Bulygen, V.V., Moran, D.M., Montiero,
F.M., Follows, M.J., Valois, F.W. & J.B. Waterbury. 2011. Iron conservation by
reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii.
Proc. Natl. Acad. Sci. 108:2184-2189.
Samuelsson, G. & Öquist, G. 1977. A method for studying photosynthetic capacities of
unicellular algae based on in vivo chlorophyll fluorescence. Physiologia Plantarum.
40:315-319.
Sanudo-Wilhelmy, S. A., Kustka, A. B., Gobler, C. J., Hutchins, D. A., Yang, M., Lwiza,
K., Burns, J., Capone, D. G., Raven, J. A. & Carpenter, E. J. 2001. Phosphorus limitation
of nitrogen fixation by Trichodesmium in the central Atlantic Ocean. Nature 411: 66-69.
Sarmiento, J. L., Slater, R., Barber, R., Bopp, L., Doney, S. C., Hirst, A. C., Kleypas, J.,
Matear, R., Mikolajewicz, U., Monfray, P., Soldatov, V., Spall, S. A. & Stouffer, R.
2004. Response of ocean ecosystems to climate warming. Global Biogeochem. Cy.
18:GB3003. doi:10.1029/2003GB002134.
Scanlan, D.J., Mann, N.H. & Carr, N.G. 1993. The response of the picoplanktonic marine
cyanobacterium Synechococcus sp. WH7803 to phosphate starvation involves a protein
homologous to the periplasmic phosphate-binding protein of Escherichia coli. Mol.
Microbial. 10:181-191
Sohm, J. A., Mahaffey, C. & Capone, D. G. 2008. Assessment of relative phosphorus
limitation of Trichodesmium spp. in the North Pacific, North Atlantic, and the north coast
of Australia. Limnol. Oceanogr. 53:2495-2502.
Sohm, J.A., Edwards, B.R., Wilson, B.G., and E. A. Webb. 2011. Constitutive
extracellular polysaccharide (EPS) production by specific isolates of Crocosphaera
watsonii. Frontiers in Microbiology. 229. doi: 10.3389/fmicb.2011.00229
Staal, M., Rabouille, S. & Stal, L. J. 2007. On the role of oxygen for nitrogen fixation in
the marine cyanobacterium Trichodesmium sp. Environ. Microbiol. 9:727-736.
Stramma, L., Johnson, G. C., Sprintal, J. & Mohrholz, V. 2008. Expanding oxygen-
minimum zones in the tropical oceans. Science 32:655-658.
153
Tortell, P. D. 2000. Evolutionary and ecological perspectives on carbon acquisition in
phytoplankton. Limnol. Oceanogr. 45:744-750.
Van Mooy, B.A.S., Rocap, G. Fredericks, H.F., Evans, C.T., Devol, A.H. 2006.
Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in
oligotrophic marine environments. PNAS. 103: 8607-8612.
Van Mooy, B.S., Fredericks, H.F., Pedler, B.E. Dyhrman, S.T., Karl, D.M., Koblížek, M.,
Lomas, M.W., Mincer, T.J., Moore, L.R., Moutin, T., Rappé, M.S., Webb, E.A. 2009.
Phytoplankton in the ocean use non-phospholipids in response to phosphorus scarcity.
Nature. 458: 69-72.
Villareal, T. A. & Carpenter, E. J. 2003. Buoyancy regulation and the potential for
vertical migration in the oceanic cyanobacterium Trichodesmium. Microb. Ecol. 45:1–10
Watkins-Brandt, K.S., Letelier, R.M., Spitz, Y.H., Church, M.J., Bottjer, D. & White,
A.E. 2011. Addition of inorganic or organic phosphorus enhances nitrogen and carbon
fixation in the oligotrophic North Pacific. Mar. Ecol. Prog. Ser. 432:17-29.
Webb, E. A., Moffett, J. W. & Waterbury J. B. 2001. Iron stress in open-ocean
cyanobacteria (Synechococcus, Trichodesmium, and Crocosphaera spp.): Identification
of the IdiA protein. Appl. Environ. Microbiol. 67:5444–5452.
Webb, E. A., Jakuba, R. W., Moffett, J. W. & Dyhrman, S. T. 2007. Molecular
assessment of phosphorus and iron physiology in Trichodesmium populations from the
western Central and western South Atlantic. Limnol. Oceanogr. 52:2221–2232.
Webb, E.A., Ehrenreich, I.M., Brown, S.L., Valois, F.W. & J. B. Waterbury. 2009.
Phenotypic and genotypic characterization of multiple strains of the diazotrophic
cyanobacterium Crocosphaera watsonii, isolated from the open ocean. Environmental
Microbiology. 11-338-348.
Wu, J. F., Sunda, W., Boyle, E. A. & Karl, D. M. 2000. Phosphate depletion in the
western North Atlantic Ocean. Science 289:759-762.
Zehr, J. P., Wyman, M., Miller, V., Duguay, L. & Capone, D. G. 1993. Modification of
the Fe protein of nitrogenase in natural populations of Trichodesmium thiebautii. Appl.
Environ. Microbiol. 59:669-676.
Zehr, J. P., Waterbury, J. B, Turner, P. J., Montoya, J. P., Omoregie, E., Steward, G. F.,
Hansen, A., & Karl, D. M. 2001. Unicellular cyanobacteria fix N
2
in the subtropical
North Pacific Ocean. Nature 412: 635-638.
154
Zehr, J. P., Bench, S. R., Mondragon, E. A., McCarren, J., & DeLong, E. F. 2007. Low
genomic diversity in tropical oceanic N
2
-fixing cyanobacteria. Proc. Nat. Acad. Sci.
1780-17812.
Zehr, J.P., Bench, S.R., Carter, B.J., Hewson, I., Niazi, F., Shi, T., Tripp, J. & Affourtit,
J.P. 2008. Globally distributed uncultivated oceanic N
2
-fixing cyanobacteria lack
oxygenic photosystem II. Science 322:1110-1112.
Abstract (if available)
Abstract
Approximately half of natural global biological dinitrogen (N2) fixation takes places in the oceans. Estimates suggest that cyanobacteria including the filamentous genus Trichodesmium and unicellular groups like Crocosphaera collectively contribute the majority of oceanic N₂ fixation. Rapidly changing environmental factors such as the rising atmospheric partial pressure of carbon dioxide (pCO₂), shallower mixed layers (higher light intensities) and changes in nutrient fluxes to the euphotic zone (from both deep water and atmospheric inputs) will likely affect N₂-fixation rates in the future ocean. Several studies using laboratory cultures of Trichodesmium erythraeum and Crocosphaera watsonii have documented increased N₂-fixation rates when pCO₂ was doubled from present-day atmospheric concentrations (~380 ppm) to 100-year projected future levels (~750 ppm). Because marine N and C biogeochemistry are tightly linked, this potential impact on the N cycle will likely have important consequences for the C cycle. These findings provided impetus for examining effects of elevated pCO₂ on N₂-fixation rates in combination with other environmental factors like iron (Fe), phosphorus (P), and light. Thus, I examined interactive effects of light and pCO₂ on growth, N₂- and CO₂-fixation rates by two strains of T. erythraeum (GBRTRLI101 and IMS101) in laboratory semi-continuous cultures. The effect of elevated pCO₂ on gross N₂-fixation rates was high in cultures (GBRTRLI101 and IMS101) growing under low (38 μmol quanta m⁻² s⁻¹) and mid irradiances (100 μmol quanta m⁻² s⁻¹), but this effect was reduced at high light (220 μmol quanta m⁻² s⁻¹). This study suggests that elevated pCO₂ may have a strong positive effect on gross N₂ fixation by Trichodesmium in intermediate and bottom layers of the euphotic zone, but perhaps not in light-saturated upper layers of the oceans. I also examined the combined effects of irradiance and pCO₂ on growth, N₂- and CO₂-fixation rates in two western tropical Atlantic Ocean isolates of C. watsonii (WH0401 and WH0402). In both strains, cellular growth, gross N₂- and CO₂-fixation rates were reduced in low-pCO₂-acclimated cultures (190 ppm) relative to present-day (~385 ppm) or future (~750 ppm) pCO₂ treatments. Unlike previous reports for C. watsonii (WH8501), however, N₂-fixation rates did not increase further in cultures acclimated to 750 ppm relative to those maintained at present-day pCO₂. Both increasing irradiance (p<0.001) and pCO₂ (p<0.03) had a significant negative effect on gross:net N₂-fixation rates in WH0402 and trends were similar in WH0401, implying that retention of fixed N was enhanced under elevated irradiance and pCO₂. These results also imply that growth rates and N2-fixation rates of WH0401 and WH0402 respond differently to changing pCO₂. These data, along with previously reported results, suggest that C. watsonii may have wide-ranging, strain-specific responses to changing irradiance and pCO₂, emphasizing the need to examine a range of isolates within this genus. In the third chapter, I examine three-way interactions between pCO₂, P availability, and irradiance in a Pacific Ocean isolate of C. watsonii (WH0003). First, I document P requirements for growth, N₂- and CO₂-fixation rates by generating Monod functional response curves under high and low pCO₂ and light conditions. The effect of elevated pCO₂ on these physiological rates was greatly enhanced under low P conditions in comparison with P-replete cultures, due to a high threshold concentration of P for these rates in low-pCO₂-acclimated cultures. This trend was consistent under low (40 μmol quanta m⁻² s⁻¹) and high (150 μmol quanta m⁻² s⁻¹) irradiance, and suggests that the P demand for growth of C. watsonii decreases with increasing pCO₂. The effect of elevated pCO₂ on N₂-fixation rates was reduced 8-fold under low light in comparison with high-light-acclimated cultures (p<0.05), reflecting a light-pCO₂ interactive trend opposite to that documented for Trichdodesmium. This difference in the interactive effect of light and pCO₂ on N₂ fixation by Crocosphaera when compared with Trichodesmium might be caused by the different N₂-fixation strategies that they use (temporal vs. spatial separation of N₂ and CO₂ fixation). These studies emphasize the need to examine interactive effects of multiple environmental variables on a variety of oceanic N₂ fixers to accurately predict effects of global change on the N and C cycles.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Iron-dependent response mechanisms of the nitrogen-fixing cyanobacterium Crocosphaera to climate change
PDF
Thermal acclimation and adaptation of key phytoplankton groups and interactions with other global change variables
PDF
The molecular adaptation of Trichodesmium to long-term CO₂-selection under multiple nutrient limitation regimes
PDF
The connection of the phosphorus cycle to diazotrophs and nitrogen fixation
PDF
B-vitamins and trace metals in the Pacific Ocean: ambient distribution and biological impacts
PDF
Spatial and temporal dynamics of marine microbial communities and their diazotrophs in the Southern California Bight
PDF
The distribution of B-vitamins in two contrasting aquatic systems, and implications for their ecological and biogeochemical roles
PDF
Future impacts of warming and other global change variables on phytoplankton communities of coastal Antarctica and California
PDF
Biological nitrogen fixation associated with living and decomposing macroalgae
PDF
The distribution and speciation of copper across different biogeochemical regimes
PDF
Oxygen uptake rates in the thermocline of coastal waters: assessing the role of carbon and inorganic nutrient inputs
PDF
The effects of heat and air pollution on mental-health related mortality
Asset Metadata
Creator
Garcia, Nathan Samuel
(author)
Core Title
Effects of global change on the physiology and biogeochemistry of the N₂-fixing cyanobacteria Trichodesmium erythraeum and Crocosphaera watsonii
School
College of Letters, Arts and Sciences
Degree
Doctor of Philosophy
Degree Program
Biology
Publication Date
05/27/2013
Defense Date
03/27/2012
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
carbon dioxide,Crocosphaera,global change,light,nitrogen fixation,OAI-PMH Harvest,phosphorus,Trichodesmium
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Hutchins, David A. (
committee chair
), Berelson, William M. (
committee member
), Capone, Douglas G. (
committee member
), Caron, David A. (
committee member
), Webb, Eric A. (
committee member
)
Creator Email
n8garcia@gmail.com,nsgarcia@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c3-121029
Unique identifier
UC11291439
Identifier
usctheses-c3-121029 (legacy record id)
Legacy Identifier
etd-GarciaNath-1357.pdf
Dmrecord
121029
Document Type
Dissertation
Rights
Garcia, Nathan Samuel
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
carbon dioxide
Crocosphaera
global change
light
nitrogen fixation
phosphorus
Trichodesmium