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The distribution of B-vitamins in two contrasting aquatic systems, and implications for their ecological and biogeochemical roles
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The distribution of B-vitamins in two contrasting aquatic systems, and implications for their ecological and biogeochemical roles
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Content
THE DISTRIBUTION OF B-VITAMINS IN TWO CONTRASTING AQUATIC
SYSTEMS, AND IMPLICATIONS FOR THEIR ECOLOGICAL AND
BIOGEOCHEMICAL ROLES
by
Laila Pualani Barada
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
(BIOLOGICAL SCIENCES)
December 2013
Copyright 2013 Laila Pualani Barada
ii
All Hail to Alma Mater
To thy glory we sing;
All Hail to Southern California
Loud let thy praises ring;
Where Western sky meets Western sea
Our college stands in majesty;
Sing our love to Alma Mater,
Hail, all hail to thee!
iii
DEDICATION
I dedicate this document to my parents, sister, and friends for their constant support,
inspiration, and continued encouragement.
iv
ACKNOWLEDGMENTS
I will be forever grateful to my mentor, Professor Capone, the other members of my
committee, and department staff. Thank you also to my family and friends, without
whom I could not have made it this far. I also thank USC for the many opportunities,
funding, and fellowship support.
v
TABLE OF CONTENTS
Epigraph ii
Dedication iii
Acknowledgments iv
List of Tables vii
List of Figures viii
Abstract xii
Introduction: The Biology of B-Vitamins
History and Ecological Relevance 1
Specific B-Vitamins 4
Thiamin 4
Pyridoxine 4
Biotin 5
Study Sites 5
The Western Tropical North Atlantic Ocean 5
Lake Tahoe, California 6
Research Objectives 7
Introduction References 7
Chapter One: The Distribution of Thiamin and Pyridoxine in the Western
Tropical North Atlantic Amazon River Plume 11
Abstract 11
Introduction 12
Materials and Methods 16
Results 19
Concentrations of B-Vitamins 19
Potential Effect of B-Vitamins on Biological Processes 22
Linear Regression Models 24
Discussion 26
Chapter One References 33
vi
Chapter Two: The Distribution and Significance of Pyridoxine and Biotin in
Lake Tahoe, California 38
Abstract 38
Introduction 39
Materials and Methods 41
Site Description 41
Sample Collection and Storage 42
Results 44
Concentrations of B-Vitamins 44
Trace Metals 47
Biological Parameters 50
Water Column Characteristics 52
Discussion 52
Chapter Two References 68
Chapter Three: Summary 73
Bibliography 78
Appendices
Appendix: Phosphonate Utilization and Methane Production in Field Populations
of Trichodesmium spp. from the Western Tropical North Atlantic
Ocean and Cultures of Trichodesmium erythraeum IMS101 89
Abstract 89
Introduction 90
Materials and Methods 93
Results 95
Discussion 103
Ecological Relevance of MnP and the Marine Methane Cycle 103
Metabolic
Efficiency
of
Different
Phosphorus
Sources
104
Preferred Source of Phosphorus 104
References 107
vii
LIST OF TABLES
Table 1-1: Table 1. Statistical test results comparing B-vitamin
concentrations at surface depths with below surface or
halocline depths. Vitamin B
1
followed by B
6
, the specific
statistical test, failed T-test assumptions, and p value for
each station is listed. 20
Table 1-2: Table 2. Correlation coefficients of vitamins B
1
and B
6
with
nitrogen and carbon fixation in the less than and greater than
10-µm size classes, direction of relationships, correlation
coefficients, and p values. 23
Table 1-3: Table 3. Multiple linear regression model factor coefficients
and statistical results for carbon fixation in the less than 10-
µm size class and nitrogen fixation in the greater than 10-µm
size class. 25
Table 1-4: Table 4. Global B-vitamin concentrations including current
and previous studies. n/a not available, n/d not detectable 28
Table 2-1: Table 1. Trace metal concentrations at various freshwater
lakes and rivers (WLT and MLT, West and Mid Lake Tahoe
stations, respectively). 57
Table 2-2: Table 2. Pearson product moment correlation analysis of
vitamins B
6
and B
7
, and trace metals at the West and Mid
Lake Tahoe stations (WLT and MLT, respectively). R =
correlation coefficient 64
viii
LIST OF FIGURES
Figure 1-1: Figure 1. Study sites in the WTNA Ocean with degrees
latitude north and degrees longitude west shown. Stations
clustered by sea surface salinity (SSS): low salinity stations
(SSS < 30, yellow circles), mesohaline stations (30 < SSS >
35, green circles) and oceanic/open ocean stations (SSS >
35, blue circles). Ocean data view (Schlitzer, R., 2011). 16
Figure 1-2: Figure 2. Depth profiles of dissolved vitamins (B
1
and B
6
)
measured in the WTNA Ocean; (A) low salinity stations;
(B) mesohaline stations; (C) oceanic/open ocean stations.
Stations are ordered by sea surface salinity (SSS) moving
from the lowest to highest SSS. Surface concentration of
vitamin B
1
for station 11 (964.8 ± 426 pM) omitted due to
concerns with possible contamination and for visualization
of variation within the depth profile (average concentrations
± 1 standard deviation). Vertical lines show the detection
limit (D/L) of vitamin B
1
(solid line) and B
6
(dashed line). 21
Figure 1-3: Figure 3. Carbon and nitrogen fixation rates in the less than
and greater than 10-micrometer size classes with B-vitamin
depth profiles; (A) Station 7; (B) Station 1; (C) Station 9.1;
(D) Station 9.1. 24
Figure 1-4: Figure 4. Multiple linear regression models. Nutrients;
phosphate: PO
4
3-
; silicate: dSi; thiamin: B
1
; Mixed layer
depth: MLD; photosynthetically active radiation: PAR;
fluorescence: fluor.; and Temperature (°C): Temp. Stations
are separated by sea surface salinity, solid line below low
salinity stations, dotted line below mesohaline stations, and
dashed line below oceanic stations, A) Carbon fixation < 10-
micrometer size class, and (B) Nitrogen fixation > 10-
micrometer size class. 25
Figure 2-1: Figure 1. Sampling locations in Lake Tahoe located on the
California and Nevada border. The two stations, Mid Lake
Tahoe (white circle next to white state border) and West
ix
Lake Tahoe are marked (white circle on the west side of the
lake, Photo credit- terc.ucdavis.edu, State of the Lake
Report, 2012). 42
Figure 2-2: Figure 2. Profiles of dissolved B-vitamin concentrations
(pM ± 1 standard deviation): pyridoxine (B
6
), biotin (B
7
),
and cobalamin (B
12
), and the amino acid methionine in the
water column at the (A) West Lake Tahoe station and (B)
Mid Lake Tahoe stations. 46
Figure 2-3: Figure 3. Profiles of dissolved (A) pyridoxine and (B) biotin
concentrations (pM, average ± 1 standard deviation) in the
water column at the West and Mid Lake Tahoe stations
(WLT and MLT, respectively). 47
Figure 2-4A: Figure 4. (A) Profiles of dissolved trace metal
concentrations (nM) in the water column at the West and
Mid Lake Tahoe stations (WLT and MLT, respectively). 49
Figure 2-4B: Figure 4. (B) Continuation: profiles of dissolved trace metal
concentrations (nM) in the water column at the West and
Mid Lake Tahoe stations (WLT and MLT, respectively). 50
Figure 2-5: Figure 5. Depth profiles of (A) Chlorophyll a (Chl a)
concentrations (µg L
-1
, average ± 1 standard deviation) and
(B) Bacterial abundance (cells ml
-1
, average ± 1 standard
deviation) in the water column at the West and Mid Lake
Tahoe stations (WLT and MLT, respectively). 51
Figure 2-6: Figure 6. Ancillary data: temperature (°C) and salinity
(PSU). Data collected from a CTD sensor at the West and
Mid Lake Tahoe stations (WLT and MLT, respectively). 52
Figure 2-7A: Figure 7. (A) Profiles of dissolved pyridoxine and biotin
(pM, average ± 1 standard deviation) with trace metal
concentrations (nM) in the water column at the West Lake
Tahoe station. 59
Figure 2-7B: Figure 7. (B) Continuation: profiles of dissolved pyridoxine
and biotin (pM, average ± 1 standard deviation) with trace
metal concentrations (nM) in the water column at the West
Lake Tahoe station. 60
x
Figure 2-8A: Figure 8. (A) Profiles of dissolved pyridoxine and biotin
(pM, average ± 1 standard deviation) and trace metal
concentrations (nM) in the water column at the Mid Lake
Tahoe station. 61
Figure 2-8B: Figure 8. (B) Continuation: profiles of dissolved pyridoxine
and biotin (pM, average ± 1 standard deviation) and trace
metal concentrations (nM) in the water column at the Mid
Lake Tahoe station. 62
Figure 2-9: Figure 9. di-Nitrogen (N
2
) and carbon (C) fixation (nmol L
-1
hr
-1
, average ± 1 standard deviation) at the West and Mid
Lake Tahoe stations (WLT and MLT, respectively). 67
Figure A-1: Figure 1. Absorbance (600 nm wavelength) of
Trichodesmium erythraeum IMS101 cultures grown in
media containing methylphosphonate, phosphate, both
methylphosphonate and phosphate, and a phosphorus
deplete control. 96
Figure A-2: Figure 2. Absorbance (600 nm wavelength) of the bacterial
community associated with Trichodesmium erythraeum
IMS101 cultures grown in media containing
methylphosphonate, phosphate, both methylphosphonate
and phosphate, and a phosphorus deplete control. 97
Figure A-3: Figure 3. Absorbance (600 nm wavelength) of cultures
inoculated with 0.2 micron filtered Trichodesmium
erythraeum IMS101 cultures grown in media containing
methylphosphonate, phosphate, both methylphosphonate
and phosphate, and a phosphorus deplete control. 98
Figure A-4: Figure 4. Methane (µM) production in Trichodesmium
erythraeum IMS101 cultures grown in media containing
methylphosphonate, phosphate, both methylphosphonate
and phosphate, and a phosphorus deplete control. 99
Figure A-5: Figure 5. Methane (µM) production in the associated
microbial
community
of
Trichodesmium erythraeum IMS101 cultures
grown in media containing methylphosphonate, phosphate,
both methylphosphonate and phosphate, and a phosphorus
deplete control. 100
xi
Figure A-6: Figure 6. Methane (µM) production in cultures inoculated
with 0.2 micron filtered Trichodesmium erythraeum IMS101
cultures grown in media containing methylphosphonate,
phosphate, both methylphosphonate and phosphate, and a
phosphorus deplete control. 100
Figure A-7: Figure 7. Absorbance (600 nm wavelength) of
Trichodesmium spp. collected from the WTNA in filtered
seawater containing methylphosphonate, phosphate, and a
phosphorus deplete control. 102
Figure A-8: Figure 8. Methane (µM) production of Trichodesmium spp.
collected from the WTNA in filtered seawater containing
methylphosphonate, phosphate, and a phosphorus deplete
control. 103
xii
ABSTRACT
B-‐vitamins
are
recognized
as
important
organic
growth
factors,
although
our
knowledge
regarding
their
concentrations
and
distribution
in
aquatic
ecosystems
is
limited.
We
present
the
first
direct
measurements
of
the
organic
growth
factors
thiamin
(B1)
and
pyridoxine
(B6) in
the
North
Atlantic
Ocean
that
is
influenced
by
Amazon
river
plume.
This
is
an
area
known
to
have
high
productivity,
di-‐nitrogen
(N2)
fixation,
and
carbon
(C)
sequestration.
The
first
directly
measured
vitamin
B6
and
biotin
(B7)
concentrations
from
an
oligotrophic
freshwater
system,
Lake
Tahoe,
are
also
presented.
B-‐vitamins
function
as
essential
enzymatic
co-‐factors
for
diverse
biological
reactions.
Specifically,
vitamins
B1
and
B7
are
involved
in
carbon
metabolism
while
vitamin
B6
is
required
for
the
metabolism
of
almost
all
amino
acids.
Therefore,
vitamins
B1,
B6,
and
B7
may
play
critical
roles
in
both
C
and
nitrogen
(N)
cycling
in
aquatic
environments
as
many
phytoplankton
cannot
synthesize
these
growth
factors
and
need
to
acquire
them
from
the
environment.
These
studies
draw
attention
to
the
potential
roles
of
B-‐vitamins
in
ecosystem
dynamics.
Concentrations
of
vitamins
B1
and
B6 in
the
WTNA
Ocean
ranged
from
undetectable
to
230
and
40
pM,
respectively.
Depth
profiles
in
the
photic
zone
of
B1
and
B6 varied
with
depth
and
salinity.
Vitamin
B1
concentrations
were
significantly
higher
in
the
surface
plume
waters
at
some
stations
suggesting
a
possible
riverine
influence.
Linear
regression
models
were
used
to
determine
the
influence
of
vitamins
B1
and
B6
on
biologically
mediated
C
and
N
fixation.
The
results
indicated
xiii
that
the
availability
of
these
co-‐enzymes
could
affect
the
rates
of
these
processes
in
the
WTNA.
Specifically,
significant
increases
in
C
and
N2
fixation
were
observed
with
increasing
concentrations
of
vitamin
B1
(low
salinity
and
mesohaline
stations
9.1
and
1,
p
value
<
0.017
and
<
0.03,
respectively).
A
significant
positive
correlation
was
also
observed
between
N2
fixation
and
vitamin
B1
at
station
1
(p
value
<
0.29)
and
vitamin
B6
at
station
9.1
(p
value
<
0.017).
This
study
suggests
that
a
dynamic
interplay
is
possible
between
these
organic
growth
factors
and
biologically
mediated
C
and
N2
fixation
that
ultimately
affect
global
biogeochemical
cycling.
Concentrations
of
vitamins
B6
in
Lake
Tahoe
ranged
from
undetectable
to
3.17
and
3.67
pM
at
the
West
Lake
Tahoe
(WLT)
and
Mid
Lake
Tahoe
(MLT)
stations
respectively.
Vitamin
B7
concentrations
ranged
from
0.59 to 4.28 pM and 0.23 to 3.45
pM at the WLT and MLT stations, respectively. Other B-vitamins were below the
detection limits suggesting that dissolved B-vitamin concentrations in the water column
were very low during this study. Generally, the WLT station had higher trace metal
concentrations compared to the MLT station suggesting a potential terrestrial source of
trace metals to the lake. Depth profiles showed corresponding peaks in trace metals and
B-vitamins, and correlation analysis showed a significant relationship of some trace
metals and B-vitamins that tended to increase together. This suggests possible trace
metal limitation or co-limitation of B-vitamin biosynthesis.
Collectively these studies highlight the importance of B-vitamins to various
aquatic systems because of their ability to affect rates of biologically mediated C and N
2
xiv
fixation, community structure, and ecosystem functioning. Multiple factors contribute to
the abundance and distribution of B-vitamins, specifically species distribution and trace
metal concentrations. However, further studies are required to determine the magnitude
of the influence of B-vitamins on global biogeochemical cycling and other factors
affecting their distribution in various aquatic habitats.
1
INTRODUCTION: THE BIOLOGY OF B-VITAMINS 1
History and Ecological Relevance 2
B-vitamins form a group of water-soluble organic growth factors that play 3
essential roles in the regulation of cellular metabolism. Eight different B-vitamins have 4
been described thus far including thiamin (B
1
), riboflavin (B
2
), niacin (B
3
), pantothenic 5
acid (B
5
), pyridoxine (B
6
), biotin (B
7
), folic acid (B
9
), and cobalamin (B
12
). B-vitamins 6
play significant roles in diverse metabolic reactions, including the most basic ones 7
required to sustain life (Snell, 1953). They function as coenzymes for many of the most 8
important enzymatic reactions in biology, including those involved in the Calvin and 9
TCA cycles, fatty acid metabolism, amino acid biosynthesis, and nucleic acid metabolism 10
(Voet et al., 2001). Our understanding of the importance of B-vitamins to aquatic 11
ecosystems continues to increase, yet further studies focusing on the sources, sinks, 12
cycling, and influences on biogeochemical cycles are needed. 13
Our knowledge of B-vitamin concentrations in aquatic systems has historically 14
been determined using indirect bioassay techniques (Carlucci and Bowes, 1972; 15
Natarajan and Dugdale, 1966). Much of our information has come from marine systems 16
as concentrations in fresh water were often below the limit of detection using the older 17
bioassay method (Burkholder and Burkholder, 1958). Due to the development of direct 18
and more sensitive B-vitamin testing techniques, our understanding has begun to 19
substantially increase (Barada et al., 2013; Sañudo-Wilhelmy et al., 2012). The new 20
testing methods have a limit of detection in the low pM range and can be utilized to study 21
the presence of the low-concentration B-vitamins in the aquatic environments (Sañudo- 22
2
Wilhelmy et al., 2012). We now have greater ability to determine the concentration, 23
distribution and subsequent influence of B-vitamins in aquatic systems. 24
Historically, the focus of investigation was centered on only three of the B- 25
vitamins, B
1
, B
7
, and B
12
. The others were generally overlooked as the importance and 26
requirements of B-vitamins for the growth of organisms was unknown (Provasoli and 27
Pintner, 1953; Droop, 1957; Burkholder and Burkholder, 1958; Carlucci and Bowes, 28
1970; 1972; Strickland, 2009). Investigations into the influence of the other B-vitamins 29
on biology began when it was discovered that one of the most abundant marine bacteria 30
species was lacking the biosynthetic genes required for the production of essential 31
vitamins (Giovannoni et al., 2005). After publication of the Pelagibacter ubique genome, 32
it was discovered that de-novo synthesis of B-vitamins was absent in bacteria belonging 33
to the SAR86 clade, which is highly abundant in the surface oceans (Dupont et al., 2011). 34
The importance of these species is demonstrated by their global distribution, biomass, 35
and productivity. Thus, it is likely that these organisms as well as other important players 36
in global biogeochemical cycles are influenced by the presence of B-vitamins in the 37
surrounding water. In fact, is has now been shown that over half of the phytoplankton 38
species investigated thus far are auxotrophs for at lease one B-vitamin (Croft et al., 39
2006). These studies suggest that exogenous sources of B-vitamins are essential for 40
many biologically important organisms that ultimately drive global biogeochemical 41
cycles. 42
The ecological relevance of B-vitamins was demonstrated when they were shown 43
to be limiting or co-limiting growth factors in some aquatic ecosystems (Panzeca et al., 44
3
2006; Bertrand et al., 2007). B-vitamin concentrations in large areas of the oceans have 45
been found to be below the limit of detection (Sañudo-Wilhelmy et al., 2012). 46
Historically, freshwater systems have been found to have even lower concentrations than 47
those measured in marine systems (Benoit, 1957; Natarajan and Dugdale, 1966). Since 48
these organic growth factors likely play a role in regulating production in all aquatic 49
systems, further studies will be required to fully understand the ecological relevance of 50
B-vitamins. 51
Freshwater inputs from groundwater and rivers have been shown to be important 52
contributors of both macronutrients and essential trace metals to the oceans (Boyle et al., 53
1982; Tovar-Sanchez and Sañudo-Wilhelmy, 2011). Recent studies have also shown an 54
inverse correlation between B-vitamin concentrations and salinity (Gobler et al., 2007) 55
that suggests freshwater inputs may be a source of B-vitamins to aquatic systems. The 56
relationship between freshwater river flow and dissolved B-vitamins from rivers into 57
coastal oceans has not previously been investigated. Our study is the first to research 58
dissolved B-vitamin concentrations in the WTNA Amazon River plume using the newer 59
more sensitive methods. 60
Freshwater intrusion from watershed runoff, rivers, and groundwater has been 61
documented to be a source of trace metals to Lake Tahoe (Goldman, 1979; Nagy, 2003). 62
These inputs of water are also a potential source of B-vitamins to the lake. The sources, 63
sinks, and cycling of B-vitamins in lakes are understudied. Ours is the first study to 64
investigate B-vitamins in Lake Tahoe using the direct method, and will help to provide a 65
foundation for future studies. 66
4
Specific B-Vitamins 67
Thiamin 68
Vitamin B
1
is an essential organic growth factor that is required by all known 69
organisms. It functions by associating with a number of enzymes involved primarily in 70
carbohydrate and branched-chain amino acid metabolism. Vitamin B
1
plays a vital role 71
by associating with the central enzymes in various biogeochemical reactions involved in 72
carbon (C) transformations (Henkes et al., 2001; Jordan, 2003; Pohl, 2004). Perhaps the 73
most striking example of this can be demonstrated by the relationship B
1
has with 74
pyruvate decarboxylase, an enzyme responsible for connecting glycolysis with the citric 75
acid cycle for the production of energy by aerobic organisms. B
1
is also known to play a 76
key role with the enzyme transketolase, which is involved in the Calvin cycle, the C 77
fixation reactions of photosynthesis, and the pentose phosphate pathway (Henkes et al., 78
2001; Jordan, 2003). The result is the generation of NADH and 5-carbon sugars. Thus, it 79
is likely that thiamin plays an important role in the global biogeochemical cycling of C, a 80
vital regulator of major processes. 81
Pyridoxine 82
Vitamin B
6
was first discovered in the 1930’s (Ohdake, 1932), which allowed 83
researchers to grow two species of the bacterium Clostridium in a defined medium for the 84
first time (McDaniel et al., 1939). They were subsequently shown to be essential for the 85
growth of some species of marine and freshwater algae as early as the 1950’s (Provasoli 86
and Pinter, 1953). B
6
is now known to function in over 160 biochemical reactions mainly 87
involving amino acids (Snell, 1953; Percudani and Peracchi, 2009). Two of these amino 88
5
acids in particular, glutamate and glutamine, are integral to the initial steps of ammonia 89
assimilation and incorporation of nitrogen (N) into amino acids. Ammonia is the product 90
of di-nitrogen (N
2
) fixation and vitamin B
6
plays an integral role in regulating this 91
important biological process. Thus, it is likely that vitamin B
6
influences the global N 92
cycle and as a consequence the global C biogeochemical cycle as well. 93
Biotin 94
Vitamin B
7
functions as a catalyst for enzymes, specifically carboxylases, 95
required for the transfer of CO
2
groups from bicarbonate to acceptor molecules, i.e. the 96
metabolism of single carbon subunits (Knowles, 1989). It is known to be able to catalyze 97
enzymatic reactions for the following classes of enzymes: ligases, lyases, and transferases 98
(Nikolau et al., 2003). These enzymes are used in many different biosynthesis pathways 99
including fatty acid synthesis (e.g. acetyl coenzyme A), mediation of sodium transport 100
(e.g. oxaloacetate), and the coupling of two-carbon carboxylations, known as 101
transcarboxylase (Alban et al., 2000; Knowles, 1989). Vitamin B
7
was found to be 102
ecologically relevant in the 1960’s, when it was discovered that is could limit or co-limit 103
phytoplankton growth and total biomass (Carlucci and Silbernagel, 1969). Therefore, it 104
is likely that this vitamin also influences global biogeochemical cycles, primarily the C 105
cycle. 106
Study Sites 107
The Western Tropical North Atlantic Ocean 108
The Amazon River flows through Brazil ending in the Western Tropical North 109
Atlantic Ocean. It is responsible for the largest influx of freshwater into any of the 110
6
world’s oceans and results in a low salinity plume covering up to 2 x 10
6
km
2
111
(Subramaniam et al., 2008). The plume contributes to phytoplankton succession leading 112
to elevated rates of N
2
and carbon (C) fixation (Wood, 1966; Carpenter et al., 1999; 113
Capone et al., 1997; Foster et al., 2007). Perhaps the most significant ecological 114
consequence of the processes enhanced by the river plume is elevated rates of carbon 115
sequestration (Subramaniam et al., 2008). Ocean productivity influences biogeochemical 116
cycles that are important for the regulation of global processes. Therefore, extensive 117
studies investigating the factors that influence rates of N
2
and C fixation have been 118
conducted. However, the influence of organic growth factors such as B-vitamins has not 119
been previously investigated. 120
Lake Tahoe, California 121
Lake Tahoe is an oligotrophic alpine lake located in the Sierra Nevada Mountain 122
range between California and Nevada, USA. It is one of the deepest lakes in the United 123
States with a maximum depth of 500 m and an average depth of 300 m. The small 124
watershed associated with Lake Tahoe results in low nutrient and productivity levels. 125
Historically, the lake was nitrogen and iron limited. It has now undergone a shift from 126
nitrogen to phosphorus limitation. This phenomenon was driven by anthropogenic 127
influences, which increased N additions into the lake, resulting in a shift to phosphorus 128
limitation of phytoplankton growth (Goldman, 1988 and 2000). The situation continues 129
to fluctuate as efforts to reduce pollution increase. Lake Tahoe is unique in that a 130
population of epilithic periphyton dominated by N
2
fixing blue-green algae were found to 131
be actively fixing N throughout the year (Reuter et al., 1986). Further studies are 132
7
required to determine the influence of B-vitamins on N
2
and C fixation and 133
biogeochemical cycling in this aquatic system. 134
Research Objectives 135
This research focused on three B-vitamins, B
1
, B
6
, and B
7
, in two contrasting 136
aquatic systems. The objectives of the research conducted in the Western Tropical North 137
Atlantic Amazon River plume, an area known to have elevated di-nitrogen fixation, 138
productivity, and carbon sequestration, were as follows: 1. To provide the first directly 139
measured concentrations of vitamins B
1
and B
7
, 2. To determine the influence of the 140
Amazon River plume on B-vitamin concentrations, and 3. To determine the influence of 141
vitamins B
1
and B
7
on the biogeochemical processes such as carbon and di-nitrogen 142
fixation. The objectives of the research conducted in Lake Tahoe, oligotrophic high 143
alpine freshwater systems were as follows: 1. To provide the first directly measured 144
concentrations of vitamins B
6
and B
7
, 2. To determine the influence of trace metals on B- 145
vitamin concentrations, and 3. To determine the influence of vitamins B
6
and B
7
on the 146
biogeochemical processes such as carbon and di-nitrogen fixation. 147
References 148
Alban, C., Job, D., Douce, R., (2000). Biotin metabolism in plants. Annu. Rev. Plant 149
Physiol. Plant Mol. Biol. 51; 17-47. 150
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6
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corresponding protein families. BMC Bioinformatics 10, 273–281. 228
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along the western boundary of the sub-tropical North Atlantic Ocean. 249
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102–123. 254
11
CHAPTER ONE: THE DISTRIBUTION OF THIAMIN AND PYRIDOXINE IN 255
THE WESTERN TROPICAL NORTH ATLANTIC OCEAN 256
Abstract 257
B-vitamins are recognized as essential organic growth factors for many 258
organisms, although little is known about their abundance and distribution in marine 259
ecosystems. Despite their metabolic functions regulating important enzymatic reactions, 260
the methodology to directly measure different B-vitamins in aquatic environments has 261
only recently been developed. Here, we present the first direct measurements of two B- 262
vitamins, thiamin (B
1
) and pyridoxine (B
6
), in the Amazon River plume-influenced 263
Western Tropical North Atlantic (WTNA) Ocean, an area known to have high 264
productivity, carbon (C) and di-nitrogen (N
2
) fixation, and C sequestration. The vitamins 265
B
1
and B
6
ranged in concentrations from undetectable to 230 pM and 40 pM, 266
respectively. Significantly higher concentrations were measured in the surface plume 267
water at some stations and variation with salinity was observed, suggesting a possible 268
riverine influence on those B-vitamins. The influences of vitamins B
1
and B
6
on 269
biogeochemical processes such as C and N
2
fixation were investigated using a linear- 270
regression model that indicated the availability of those organic factors could affect these 271
rates in the WTNA. In fact, significant increases in C fixation and N
2
fixation were 272
observed with increasing vitamin B
1
concentrations at some low and mesohaline stations 273
(stations 9.1 and 1; p value <0.017 and <0.03, respectively). N
2
fixation was also found 274
to have a significant positive correlation with B
1
concentrations at station 1 (p value = 275
12
0.029), as well as vitamin B
6
at station 9.1 (p value <0.017). This work suggests that 276
there can be a dynamic interplay between essential biogeochemical rates (C and N
2
277
fixation) and B-vitamins, drawing attention to potential roles of B-vitamins in ecosystem 278
dynamics, community structure, and global biogeochemistry. 279
Introduction 280
The Amazon River has the largest freshwater discharge of any river into the world 281
ocean. This results in an influx of low-salinity, nutrient-rich water into the Western 282
Tropical North Atlantic (WTNA) Ocean (Subramaniam et al., 2008). The environmental 283
conditions resulting from the river plume, influencing approximately 2 million km
2
in the 284
WTNA, contributes to phytoplankton species succession, high rates of primary 285
production, and a significant carbon (C) sink (Subramaniam et al., 2008). The neritic, 286
high-nutrient areas of the plume are dominated by diatoms that utilize the nitrogen (N) 287
and silicate (Si) supplied by the river leading to nutrient depletion in the water column. 288
Following nutrient draw down, a shift in community composition occurs beginning with 289
diatom-diazotroph associations (DDAs) as N becomes limited but sufficient supplies of 290
Si are still present. This is followed by a subsequent community shift to more typical 291
oceanic N
2
fixing organisms such as Trichodesmium spp. (Wood, 1966; Carpenter et al., 292
1999; Capone et al., 1997; Foster et al., 2007). The succession of phytoplankton species 293
supports an extensive area of increased C and di-nitrogen (N
2
) fixation resulting in a C 294
sink of approximately 1.7 Tmol annually (Subramaniam et al., 2008). Although many of 295
the factors that limit C and N
2
fixation in this region have been extensively studied, the 296
roles of organic growth factors such as B-vitamins have not been investigated despite 297
13
their biological importance. With recent advances in analytical methodologies that 298
directly measure B-vitamins in marine systems (Sañudo-Wilhelmy et al., 2012), we can 299
now start understanding the processes influencing the distribution and concentrations of 300
B-vitamins in the world ocean. River and groundwater inputs are thought to be sources 301
of B-vitamins as their concentrations have been inversely correlated with salinity (Gobler 302
et al., 2007) and river plumes have previously been shown to transport macronutrients 303
and trace metals to the ocean (Boyle et al., 1982; Tovar-Sanchez and Sañudo-Wilhelmy, 304
2011). However, the transport of dissolved B-vitamins from rivers to the coastal ocean 305
has never been evaluated. This study represents the first attempt to establish the 306
importance of the Amazon River as a source of some B-vitamins to the WTNA Ocean.
307
B-vitamins are essential coenzymes for many diverse biochemical reactions, 308
including enzymes in the Calvin cycle, amino acid biosynthesis, the TCA cycle, and 309
nucleic acid metabolism (Voet et al., 2001). Fitting with their central role in metabolism, 310
B-vitamins were recognized as important promoters of bacterial growth as early as the 311
1930s (McDaniel et al., 1939) and by the 1950s were found to be essential for the 312
cultivation of many marine and freshwater algae (Provasoli and Pinter, 1953). Recent 313
studies have confirmed the ecological relevance of B-vitamins in the environment by 314
demonstrating their ability to limit or co-limit phytoplankton growth and biomass 315
(Panzeca et al., 2006; Bertrand et al., 2007), including harmful algal blooms (Tang et al., 316
2010). Furthermore, Sañudo-Wilhelmy et al. (2012) recently showed that large areas of 317
the ocean are vitamin depleted. However, no study has addressed the influence of some 318
B-vitamins on C and N
2
fixation in the Atlantic Ocean, and herein we describe the 319
14
potential relationship between two B-vitamins, thiamin (B
1
) and pyridoxine (B
6
), 320
concentrations and biogeochemical rates in the Amazon-influenced WTNA Ocean.
321
Vitamin B
1
is an essential organic growth factor required by most organisms and 322
plays an integral role in biogeochemical reactions involving C transformations (Henkes et 323
al., 2001; Jordan, 2003; Pohl, 2004). It functions by associating with a number of 324
important enzymes including pyruvate dehydrogenase, which bridges glycolysis and the 325
citric acid cycle, as well as transketolase, which plays a critical role in the Calvin cycle 326
(C fixation reactions of photosynthesis) and the pentose phosphate pathway (Henkes et 327
al., 2001; Jordan, 2003). Many bacteria and Protista have been shown to require 328
vitamins. 329
Vitamin B
6
was first identified in 1932 by Ohdake, and is now known to catalyze 330
over 160 biochemical reactions that mainly involve amino acid transformations (Snell, 331
1953; Percudani and Peracchi, 2009). Because the role that the amino acids glutamine 332
and glutamate have in the assimilation of ammonia (NH
3
), the product of N
2
fixation, 333
which is incorporated into two amino acids (Staley et al., 2007), we hypothesized that 334
vitamin B
6
concentrations and availability could therefore also influence the N cycle. 335
Previous field and laboratory studies have focused on the vitamins B
1
, B
7
, and B
12
336
as they were thought to be required for growth, while other B-vitamins (e.g., B
6
) were 337
largely ignored (Provasoli and Pintner, 1953; Droop, 1957; Burkholder and Burkholder, 338
1958; Carlucci and Bowes, 1970; 1972; Strickland, 2009). This paradigm shifted when 339
the genome of one of the most abundant bacteria in the ocean, Pelagibacter ubique, was 340
first published revealing the absence of the genes required for the biosynthetic pathways 341
15
of vitamins B
1
and B
6
(Giovannoni et al., 2005). P. ubique belongs to the SAR11 clade, 342
which accounts for a third of all heterotrophic cells present in surface waters (Morris et 343
al., 2002), and thus plays a large role in the global C cycle. Subsequently, the genes 344
required for the de-novo synthesis of B-vitamins were found to be absent from bacteria 345
belonging to the SAR86 clade, which are highly abundant uncultured members of marine 346
surface bacterial populations (Dupont et al., 2011). In fact, over half of marine 347
phytoplanktonic species investigated thus far are auxotrophic, which includes some of the 348
most abundant and ubiquitous marine species (Croft et al., 2006), highlighting the 349
importance of external sources of B-vitamins, including vitamin B
1
. These genomic data 350
suggest that exogenous B-vitamin pools are essential for the survival of some marine 351
plankton, as they rely solely on the environment to meet their B-vitamin requirements. 352
The availability of vitamins B
1
and B
6
may therefore play a significant role in N and C 353
cycling, and may be previously unknown factors contributing to the regulation of the 354
“biological carbon pump.” However, little is known about the sources and sinks of B- 355
vitamins in marine systems or how they cycle between vitamin producers and consumers. 356
Despite the biologically important role vitamins B
1
and B
6
play in ecologically 357
relevant enzymes involved in C and N cycling, primarily carbohydrate and amino acid 358
metabolism, little is known about their concentrations or distributions in marine systems. 359
The objectives of this study were: 1) to provide the first directly measured depth-profiles 360
of vitamins B
1
and B
6
in a highly productive region of the WTNA, 2) to determine the 361
spatial distributions of those vitamins in that region, 3) to determine the influence of the 362
16
Amazon River Plume on that spatial gradient, and 4) to determine the importance of these 363
vitamins in C and N cycles. 364
Materials and Methods 365
Samples were collected in the WTNA on board the R/V Knorr as part of the 366
Amazon influence on the Atlantic: carbon export from nitrogen fixation by diatom 367
symbioses (ANACONDAS) project from May 23 to June 22, 2010. Sampling stations 368
were located between Longitude 59°67’E and 45°01’E and Latitude 4°00’N and 12°98’N 369
(Figure 1). Stations were grouped by sea surface salinity (SSS) and designated as low 370
salinity (SSS < 30, stations 4, 9.1, 10, and 11), mesohaline (30 < SSS > 35, stations 1-3, 371
and 9), and oceanic (SSS > 35, stations 7, 8, and 27). 372
Figure
1-‐1.
Study
sites
in
the
WTNA
Ocean
with
degrees
latitude
north
and
degrees
longitude
373
west
shown.
Stations
clustered
by
sea
surface
salinity
(SSS):
low
salinity
stations
(SSS
<
30,
374
yellow
circles),
mesohaline
stations
(30
<
SSS
>
35,
green
circles)
and
oceanic/open
ocean
375
stations
(SSS
>
35,
blue
circles).
Ocean
data
view
(Schlitzer,
R.,
2011). 376
Vitamin samples were collected from the top 150 meters using a Niskin Bottle 377
Rosette sampler and filtered through a 0.2-µm Supor filter (PALL, Life Sciences) using a 378
17
peristaltic pump. The filtrate was collected in 250 ml acid cleaned high-density 379
polyethylene bottles and frozen until analysis. Vitamin samples were extracted and pre- 380
concentrated according to the method of Sañudo-Wilhelmy et al., (2012). Briefly, 381
samples were passed through solid-phase C18 resin at a flow rate of 1 ml min
-1
to 382
concentrate vitamins. Samples were adjusted to pH 6.5 before being passed through the 383
resin, and then adjusted to pH 2.0 to obtain maximum vitamin recovery. Vitamins were 384
subsequently eluted off the columns with methanol, dried, and dissolved in 200 µl of 385
MilliQ water. Vitamin concentrations were then quantified using liquid 386
chromatography/tandem mass spectrometry (LC/MS/MS). Each extraction included a 387
blank and spiked positive control to test for contamination and extraction efficiency. 388
Detection limit of vitamins B
1
and B
6
were 0.81 pM and 0.61 pM, respectively (Sañudo- 389
Wilhelmy et al., 2012). Some controls used for estimating extraction efficiency with a 390
vitamin spike were compromised by vitamin-contaminated DI water yielding in some 391
cases efficiency greater than 100%. However, for most of the samples, extraction 392
efficiency was close to 100%. 393
Chlorophyll a (Chl a) samples were collected from a Niskin Bottle Rosette into 1 394
L amber bottles, filtered onto 25 mm GF/F filters and analyzed according to the EPA 395
modified fluorometric method 445.0 (Arar and Collins, 1997) in a Turner Designs 396
Trilogy Fluorometer. Sample volumes ranged from 500 mL to 1 L depending on 397
biomass. In general, oceanic stations utilized 1 L volumes, while mesohaline and low 398
salinity stations had higher biomass allowing only 500 mL volumes to be filtered. 399
18
N
2
fixation and C fixation were performed according to the method of Montoya et 400
al., (1996, 2006) using 4 L polycarbonate bottles completely filled and equipped with 401
silicone rubber caps. Bottles were enriched with 3 ml of 99%
15
N
2
(Isotec) and 250 µL of 402
0.1 M NaH
13
CO
3
(Sigma). After on-deck incubation for 24 h at surface seawater 403
temperature and simulated conditions of light for the collection depth, bottles were pre- 404
filtered through 10-micrometer Nitex mesh onto pre-combusted GF/F filters. Material on 405
the 10-micrometer filter was washed onto GF/F filters. Filters were dried and stored until 406
mass spectrometric analysis in the laboratory. Isotope abundances were measured by 407
continuous-flow isotope ratio mass spectrometry using a CE NA2500 elemental analyzer 408
interfaced to a Micromass Optima mass spectrometer. 409
Statistical analysis was performed using SigmaPlot’s (Systat Software Inc.) T-test 410
except when assumptions of normality and equal variance were violated resulting in the 411
use of the non-parametric Mann-Whitney rank sum to test for identical distributions. The 412
degree to which C and N
2
fixation correlated with each of the B-vitamins was evaluated 413
by means of a Pearson product moment correlation test. Linear regression models were 414
performed using R v2.12.2 statistical programming language (R development core, 415
2012). Exhaustive step-wise general linear regression models and leave one out cross 416
validation for generalized linear models utilized the following packages: boot (Canty and 417
Ripley, 2012), leaps (Lumley and Miller, 2009), randomForest (Liaw and Wiener, 2002), 418
and Data Analysis and Graphics (Maindonald and Braun, 2012). Due to missing data, the 419
parameters omitted from this analysis were PAR, Chl a, and cell counts. 420
421
19
Results 422
Concentrations of B-Vitamins 423
Vitamin B
1
in the WTNA varied widely among stations and ranged from 424
undetectable to 229 pM (Figure 2), except for the surface sample at station 11 measuring 425
964 pM and was suspected to be compromised by sample contamination. The lowest 426
concentrations of vitamin B
1
were measured at the oceanic stations (undetectable to 50 427
pM) followed by low salinity stations (2.5 to 184 pM), and the highest concentrations 428
were observed at mesohaline stations (undetectable to 229 pM, Figure 2). Vitamin B
6
429
concentrations also varied widely among stations ranging from undetectable to 36 pM. 430
B
6
concentrations were lowest at the mesohaline stations (undetectable to 7 pM) followed 431
by oceanic stations (undetectable to 20 pM), and were highest at low salinity stations 432
(undetectable to 36 pM, Figure 2). In general, higher concentrations of B-vitamins were 433
found at lower salinity stations and were significantly higher in the surface plume water 434
at some stations suggesting a riverine source (Table 1). There was no clear spatial trend 435
observed between the two vitamins suggesting they function and behave differently from 436
one another, and the high variability suggests a dynamic behavior influenced by sources 437
and sinks. 438
439
20
Table
1-‐1.
Statistical
test
results
comparing
B-‐vitamin
concentrations
at
surface
440
depths
with
below
surface
or
halocline
depths.
Vitamin
B1
followed
by
B6,
the
specific
441
statistical
test,
failed
T-‐test
assumptions,
and
p
value
for
each
station
is
listed.
442
443
21
Figure
1-‐2.
Depth
profiles
of
dissolved
vitamins
(B1
and
B6)
measured
in
the
WTNA
Ocean;
(A)
444
low
salinity
stations;
(B)
mesohaline
stations;
(C)
oceanic/open
ocean
stations.
Stations
are
445
ordered
by
sea
surface
salinity
(SSS)
moving
from
the
lowest
to
highest
SSS.
Surface
446
concentration
of
vitamin
B1
for
station
11
(964.8
±
426
pM)
omitted
due
to
concerns
with
447
possible
contamination
and
for
visualization
of
variation
within
the
depth
profile
(average
448
concentrations
±
1
standard
deviation).
Vertical
lines
show
the
detection
limit
(D/L)
of
449
vitamin
B1
(solid
line)
and
B6
(dashed
line). 450
22
Potential Effect of B-Vitamins on Biological Processes 451
N
2
fixation rates were positively correlated with vitamin B
1
concentrations at 452
station 7, 8 (in the small size class), 9.1, and 10 (Table 2). N
2
fixation rates were 453
inversely correlated with vitamin B
1
at stations 1, 4, and 8 (in the large size fraction, 454
Table 2). N
2
fixation rates were positively correlated to vitamin B
6
concentrations at 455
station 1 (in the small size fraction), 8, 9.1, and 10 (in the large size fraction, Table 2). 456
N
2
fixation rates were negatively correlated to vitamin B
6
at stations 1 (in the large size 457
fraction), 4, 7, and 10 (in the small size fraction, Table 2). However, significant 458
relationships between increases in N
2
fixation rates and vitamin B
1
concentrations were 459
only observed at station 7 in the small size class (p value = 0.045, Figure 3A). A 460
significant inverse relationship was observed at station 1 in the large size fraction (p 461
value = 0.029, Figure 3B). Significant relationships between increases in N
2
fixation 462
rates and vitamin B
6
concentrations were only observed at station 9.1 in the large size 463
class (p value = 0.017, Figure 3C). 464
465
23
Table
1-‐2.
Correlation
coefficients
of
vitamins
B1
and
B6
with
nitrogen
and
carbon
466
fixation
in
the
less
than
and
greater
than
10-‐μm
size
classes,
direction
of
relationships,
467
correlation
coefficients,
and
p
values. 468
469
Carbon fixation rates were positively correlated with vitamin B
1
at stations 1 (in 470
the small size fraction), 4, 7, 9.1, and 10 (Table 2). Carbon fixation rates were inversely 471
correlated to vitamin B
1
at stations 1 (in the large size class) and 8 (Table 2). Carbon 472
fixation rates were positively correlated with vitamin B
6
at stations 1, 9.1, and 10 (Table 473
2). Carbon fixation rates were inversely correlated with vitamin B
6
at stations 4, 7, and 8 474
(Table 2). However, significant increases in C fixation rates with increasing B
1
475
concentrations were only observed at station 9.1 in both size classes (p values = 0.000008 476
and 0.004, respectively, Figure 3D). No significant relationships between vitamin B
6
477
concentrations and rates of C fixation were observed. 478
24
479
Figure
1-‐3.
Carbon
and
nitrogen
fixation
rates
in
the
less
than
and
greater
than
10-‐ 480
micrometer
size
classes
with
B-‐vitamin
depth
profiles;
(A)
Station
7;
(B)
Station
1;
(C)
Station
481
9.1;
(D)
Station
9.1.
482
Linear Regression Models 483
Linear regression models included data from all stations except station 9 where N
2
484
and C fixation data were not available. Tests were performed omitting Chl a, PAR, 485
25
and/or cell counts due to missing data. The linear model showed that the factors 486
correlating with C fixation in the small size class included Si, vitamin B
1
, and water 487
temperature (Figure 4). The model was significant with a p value of 8.83 x 10
-11
, 488
predictive error (the average deviation between the known values and the models 489
predicted values) of 721, and an R
2
value of 0.522 (Table 3). The model predicting N
2
490
fixation in the larger size class showed the most important factors were temperature, 491
MLD, and vitamin B
6
(Figure 4). The model was significant with a p value of 3.92 x 10
-
492
4
, predictive error of 6.7 x 10
-4
, and an R
2
value of 0.241 (Table 3). 493
Table
1-‐3.
Multiple
linear
regression
model
factor
coefficients
and
statistical
results
for
494
carbon
fixation
in
the
less
than
10-‐μm
size
class
and
nitrogen
fixation
in
the
greater
than
10-‐ 495
μm
size
class.
496
497
26
498
Figure
1-‐4.
Multiple
linear
regression
models.
Nutrients;
phosphate:
PO4
3-‐
;
silicate:
dSi;
499
thiamin:
B1;
Mixed
layer
depth:
MLD;
photosynthetically
active
radiation:
PAR;
fluorescence:
500
fluor.;
and
Temperature
(°C):
Temp.
Stations
are
separated
by
sea
surface
salinity,
solid
line
501
below
low
salinity
stations,
dotted
line
below
mesohaline
stations,
and
dashed
line
below
502
oceanic
stations,
A)
Carbon
fixation
<
10-‐micrometer
size
class,
and
(B)
Nitrogen
fixation
>
10-‐ 503
micrometer
size
class. 504
Discussion
505
This is the first study in the WTNA euphotic zone within the influence of the 506
Amazon River plume to directly measure the vitamins B
1
and B
6
. We observed high 507
variability in the concentrations and distributions of these vitamins in the area of study. 508
Vitamin B
1
was found below the limit of detection at mesohaline station 3 (31 and 51 m) 509
27
and oceanic station 8 (2, 10, and 100 m), and vitamin B
6
was found to be below the limit 510
of detection at low salinity station 4 (8 m), mesohaline stations 3 and 9 (51 and 100 m, 511
respectively) and oceanic station 8 (10 m). The low concentrations of B-vitamins and 512
high spatial variability observed were consistent with previous studies. In fact, in large 513
regions of the Eastern Pacific Ocean between 24°N and 34°N, B-vitamins were found to 514
be below the limit of detection (Sañudo-Wilhelmy et al., 2012). The ranges of vitamin B
1
515
concentrations measured in this study (0.05 to ~1000 pM) are consistent with previously 516
published results from both bioassays and direct measurements (Table 4). The 517
concentration of B
1
measured using bioassays ranged from 15-1633 pM in the Pacific 518
Ocean (Eppley et al., 1972; Natarajan, 1970; Natarajan and Dugdale, 1966). Direct 519
measurements of vitamin B
1
ranged from 0.7 to 600 pM in the North Atlantic Ocean 520
(Okabamichael and Sañudo-Wilhelmy, 2005; Panzeca et al., 2008), and from 521
undetectable to 500 pM in the Pacific Ocean (Sañudo-Wilhelmy et al., 2012). Vitamin 522
B
6
concentrations in the WTNA ranged from undetectable to 40 pM and were generally 523
lower than previous measurements from the North Pacific Ocean (40 to 386 pM, Sañudo- 524
Wilhelmy et al., 2012). However, they fell within the range measured in the Pacific 525
Ocean (3 to 180 pM, Sañudo-Wilhelmy et al., 2012). In summary, the concentrations of 526
B-vitamins observed in this study were consistent with previous results showing they 527
vary spatially, and are often found below the limit of detection. 528
529
28
Table
1-‐4.
Global
B-‐vitamin
concentrations
including
current
and
previous
studies.
n/a
not
530
available,
n/d
not
detectable
531
532
Recent studies on the role that B-vitamins play in marine ecosystems have shown 533
that they can limit or co-limit primary production (Panzeca et al., 2006; Bertrand et al., 534
2007; Gobler et al., 2007; Panzeca et al., 2008; Tang et al., 2010). Although this study 535
did not directly investigate the effects of vitamin additions on biological processes, some 536
conclusions can be drawn from the correlations between vitamin concentrations and rates 537
of N
2
and C fixation. This study found a significant increase in C fixation with 538
increasing ambient B
1
concentrations at low salinity station 9.1 in both size classes (p 539
value ≤ 0.004, Table 2). At low salinity station 4, the lack of correlation between C 540
fixation and B
1
concentrations could be explained by the high abundance of the diatom 541
Coscinodiscus sp. Based on isolates that have been studied it appears that this diatom 542
species does not require vitamin B
1
(Croft et al., 2006) and likely contributed to the 543
majority of C fixation at this station. Significant increases in N
2
fixation were also found 544
with increasing B
1
in the large size class at station 1 (p value = 0.029) and oceanic station 545
29
7 in the small size class (p value < 0.045). These data suggest that vitamin B
1
may be 546
limiting or co-limiting N
2
fixation in some areas of the WTNA. However, other factors 547
in parallel could also be limiting or co-limiting biological rates of N
2
fixation since low 548
PO
4
3-
concentrations were also measured at station 7, and PO
4
3-
has been previously 549
shown to limit N
2
fixation (Sañudo-Wilhelmy et al., 2001; Mills et al., 2004; Webb et al., 550
2007; Moutin et al., 2008; Van Mooy et al., 2009). Hence, vitamin B
1
appears to be 551
playing a role in C and N
2
fixation in both riverine influenced and open ocean stations. 552
These results are consistent with the role of B
1
in C metabolism but the role B
1
plays in N 553
metabolism is less clear. However, pyruvate-ferredoxin oxidoreductase, an enzyme 554
crucial for electron transfer to nitrogenase, requires thiamin (Brostedt and Nordlund, 555
1991; Bothe et al., 2010) and some diazotrophs have been shown to be B
1
auxotrophs, 556
suggesting that B
1
availability in the environment may be limiting the N biogeochemical 557
cycle. N
2
fixation was found to increase with increasing vitamin B
1
at one low salinity 558
station; however, this was not observed at other stations. Therefore, further 559
investigations such as vitamin addition experiments, which show an increase of N
2
560
fixation with B
1
amendments, are required to fully understand the role of this vitamin in 561
the WTNA N and C cycles. However, the tight correlation between B
1
and C fixation 562
observed at station 9.1 (Figure 3D) suggests that this vitamin may also be important for C 563
fixation in the WTNA, and argues for further study. 564
N
2
fixation co-varied with vitamin B
6
at low salinity station 9.1; significant 565
positive relationships were found in the larger size class between vitamin B
6
and N
2
566
fixation (p value < 0.017). However, there was not a significant relationship between N
2
567
30
fixation and concentrations of vitamin B
6
at the other stations. No significant 568
relationships were observed between C fixation and vitamin B
6
concentrations at any 569
stations. Independence of vitamin B
6
and N
2
fixation can be explained by other factors; 570
for instance, at station 7, low nutrient concentrations were observed and dissolved P may 571
have limited N
2
fixation, while station 4 was dominated with the diatom Coscinodiscus 572
sp. whose requirements for B
6
are currently unknown. Thus, at some stations N
2
fixation 573
appears dependent on B-vitamins, which appears to be limiting or co-limiting 574
biogeochemical cycles in the WTNA. Since few correlations between vitamin 575
concentration and rate measurements were observed, standing concentrations may be a 576
poor measure, auxotrophic phytoplankton may not be commonly abundant, or they are 577
getting their vitamins through symbiosis (Croft et al., 2005). However, to determine the 578
extent that N and C cycles are actually dependent on vitamin B
6
, more extensive studies 579
including vitamin addition experiments will be required. 580
Multiple linear regression models were used to identify the environmental 581
variables that correlated with biogeochemical cycles in the WTNA Ocean during our 582
study. Variables correlating to C fixation in the small size class included Si, vitamin B
1
, 583
and temperature. Two of these variables, Si and water temperature, were also identified 584
as factors affecting the distribution of N
2
and C fixing organisms in previous studies 585
(Coles and Hood, 2007; Foster et al., 2007; Webb et al., 2007; Sohm and Capone, 2008; 586
Hynes et al., 2009; Van Mooy et al., 2009; Sohm et al., 2011a; 2011b). Model results 587
were consistent with the role that vitamin B
1
plays in the Calvin cycle and C metabolism 588
(Natarajan, 1970; Jordan, 2003). Our analysis showed that temperature, MLD, and 589
31
vitamin B
6
correlated to N
2
fixation in the greater size fraction. Measured N
2
fixation 590
rates were on average an order of magnitude less than modeled rates except at depths 591
where the highest rates of N
2
fixation were measured. When the highest rates of N
2
592
fixation were observed, measured rates were an order of magnitude greater than the 593
modeled rates (Figure 4). This pattern was observed across all station types and resulted 594
in the models low R
2
value. However, this is consistent with the role vitamin B
6
plays in 595
catalyzing many diverse amino acid transformations (Percudani and Peracchi, 2009), 596
specifically with the assimilation of NH
3
into the amino acids glutamine and glutamate. 597
Collectively, these results suggest that vitamin B
1
and B
6
could be important organic 598
growth factors affecting biologically mediated C and N
2
fixation in the WTNA Ocean. 599
Insights into the potential ecological importance of B-vitamins have been 600
investigated by determining half-saturation constants (K
s
) for maximal growth for 601
vitamins B
1
and B
12
for some phytoplankton species (Tang et al., 2010). However, the K
s
602
for diazotrophic microorganisms and B-vitamins have yet to be determined. The K
s
of 603
maximal growth rates for different phytoplankton species for vitamin B
1
ranged from 6 to 604
184 pM. Some of our measured concentrations of B
1
were below the K
s
suggesting that 605
vitamin B
1
may be a limiting growth factor in the WTNA. Additional studies are needed 606
to determine the K
s
for maximal growth on different B-vitamins of endemic WTNA 607
plankton species, which will help to establish the ecological framework and importance 608
of directly measured environmental B-vitamin concentrations. 609
The influence of the Amazon River plume on B-vitamin concentrations and the 610
sources of B-vitamins in the WTNA are still unresolved. Although it has been 611
32
hypothesized that fresh water inputs from rivers and groundwater can be a source of B- 612
vitamins to marine systems (Gobler et al., 2007), clear patterns were not observed to 613
support this in the WTNA Ocean. As a general trend, there was an increase in B- 614
vitamins as salinity decreased but no linear relationship was observed, suggesting that 615
mixing of river and seawater did not solely control it. An inverse correlation was 616
observed with vitamin B
1
concentration and sea surface salinity (R value = 0.25, data not 617
shown), but no correlation was observed between vitamin B
6
and sea surface salinity (R 618
value = 0.002, data not shown). The surface water sampled during this cruise was 619
estimated to be nearly 30 days out from the mouth of the river, and may explain the weak 620
correlations found between sea surface salinity and B-vitamin concentrations. Further 621
studies investigating B-vitamin concentrations near the discharge point of the Amazon 622
River should help resolve whether the river is a source of vitamins to the WTNA. In 623
addition, the removal processes of B-vitamins are poorly understood, and the half-life of 624
these vitamins has yet to be determined. However, the half-life of some vitamins (B
1
and 625
B
12
) in seawater has been shown to occur on time scales from days to weeks (Gold et al., 626
1966; Carlucci et al., 1969), suggesting that they are highly dynamic and that local 627
production may be an important biologically available source of B-vitamins. Our 628
understanding of the ecological importance of B-vitamins in marine systems is 629
continuing to increase, with the current study demonstrating that B-vitamins are highly 630
variable and could significantly influence both N
2
and C fixation in the WTNA Ocean. 631
However, further studies are needed to determine the sources, sinks, and cycling of B- 632
vitamins in oceanographic sensitive marine systems, such as the WTNA. 633
33
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New Jersey: Wiley. 449-450. 791
Webb, E. A., Jakuba, R. W., Moffett, J. W., Dyhrman, S. T. (2007). Molecular 792
assessment of phosphorus and iron physiology in Trichodesmium populations from 793
the western Central and western South Atlantic. Limnol. Oceanogr. 52, 2221-2232. 794
Wood, F. E. J. (1966). A phytoplankton study of the Amazon region. Bull. Mar. Sci. 16, 795
102–123. 796
38
CHAPTER TWO: THE DISTRIBUTION AND SIGNIFICANCE OF 797
PYRIDOXINE AND BIOTIN IN LAKE TAHOE, CALIFORNIA 798
Abstract 799
B-vitamins are essential co-factors for enzymes for universally important 800
biological reactions. Historically, concentrations in freshwater have been found to be 801
below detection limits (BDL) using indirect methods. This study is the first to utilize a 802
newly developed direct method, liquid chromatography/tandem mass spectrometry, to 803
determine the concentrations of B-vitamins in an oligotrophic alpine freshwater system, 804
Lake Tahoe. The results showed some B-vitamins (B
1
, B
2
, B
12
, and the amino acid 805
methionine) continue to be BDL, suggesting that the concentrations of these B-vitamins 806
were very low in the water column during this study. In contrast, vitamins B
6
807
(pyridoxine) and B
7
(biotin) were found in measurable concentrations in Lake Tahoe 808
ranging from BDL to 3.67 pM and 4.3 pM at the West and Mid Lake Tahoe stations 809
respectively (WLT and MLT). In general, higher concentrations of trace metals were 810
measured at WLT compared to the MLT station. This suggests a potential terrestrial 811
source of B-vitamins to the lake, specifically at near shore locations. Peaks in trace metal 812
concentrations were found to correspond to vitamin B
6
and B
7
peaks. Correlation 813
analysis showed some trace metal and vitamin concentrations tended to increase together. 814
Specifically, higher measured B-vitamin concentrations corresponding to higher trace 815
metal concentrations were observed at the WLT station. This may be indicative of 816
freshwater B-vitamin and trace metal co-limitation of biological processes. 817
818
39
Introduction 819
B-vitamins form a group of water-soluble organic growth factors that play 820
essential roles in the regulation of cellular metabolism. They function as coenzymes for 821
many of the most important enzymatic reactions in biology, regulating metabolic 822
reactions involved in the Calvin and TCA cycles, amino acid biosynthesis and nucleic 823
acid metabolism (Croft et al., 2006). B-vitamin concentrations in aquatic systems have 824
previously been determined using indirect biological assays (Carlucci and Bowes, 1972, 825
Eppley et al., 1972; Natarajan and Dugdale, 1966; Natarajan, 1970). However, 826
concentrations of B-vitamins were mostly below the limit of detection in lakes. With the 827
recent development in methodology to directly measure B-vitamins in seawater (Sañudo- 828
Wilhelmy et al., 2012), we can now apply this method to freshwater systems. 829
Concentrations of B-vitamins that have been measured in freshwater are an order of 830
magnitude lower than seawater. This suggests that B-vitamin limitation may be more 831
likely in terrestrial lakes and streams. 832
Pyridoxine (B
6
) is involved in hundreds of biochemical reactions that are central 833
to amino acid biosynthesis (Percudani and Peracchi, 2009). B
6
was first discovered by 834
Ohdake (1932) and subsequently was found to play a significant role in diverse basic 835
metabolic reactions (Snell, 1953). The process of di-nitrogen (N
2
) fixation indirectly 836
requires vitamin B
6
and results in the production of ammonia (NH
3
). When this ammonia 837
is then assimilated into the amino acids glutamate and glutamine (Staley et al., 2007). 838
Therefore, vitamin B
6
is thought to be important in the global nitrogen (N) cycle and 839
subsequently carbon (C) cycle. Vitamin B
6
concentrations have been determined in 840
40
different marine systems (Barada et al., 2013; Sañudo-Wilhelmy et al., 2012). However, 841
vitamin B
6
in freshwater systems has not previously been investigated making this a 842
novel study with some of the first direct measurements from an oligotrophic lake. 843
Biotin (B
7
) is an essential B-vitamin required in the fatty acid biosynthesis 844
pathway (acetyl coenzyme A, CoA) and is responsible for transferring CO
2
groups from 845
bicarbonate to acceptor substrates (Alban et al., 2000). The cofactor is a CO
2
carrier 846
involved mainly in the enzymatic reactions that involve ligases, lyases, and transferases 847
(Knowles, 1989; Nikolau et al., 2003). For example, biotin is utilized by pyruvate 848
carboxylase, which is responsible for enzymatic activity regulating the conversion of 849
pyruvate to oxaloacetate during gluconeogenesis. Biotin is a sulfur-containing B-vitamin 850
and requires iron for its synthesis (Berkovitch et al., 2004), suggesting that both sulfur 851
and iron availability may be limiting factors for vitamin B
7
production in freshwater 852
systems. The ecological relevance of B-vitamins has been known since the 1930’s, and 853
specifically, biotin was shown to be able to limit or co-limit phytoplankton growth and 854
biomass (Carlucci and Silbernagel, 1969). 855
Much of our knowledge about environmental B-vitamins has come from marine 856
systems as concentrations in freshwater were often BDL for the older bioassay technique. 857
This is the first study to directly measure B-vitamin concentrations in Lake Tahoe and 858
will serve as a baseline to provide background information to further our understanding 859
of the influences of B-vitamins on species composition, aquatic production, and 860
biogeochemical cycling in freshwater systems. Expanding our knowledge of the 861
potential of B-vitamins to regulate C and N
2
fixation will further our understanding of 862
41
limitations of aquatic biogeochemical processes today and improve our ability to predict 863
them as future environmental conditions continue to fluctuate. 864
Materials and Methods 865
Site Description 866
Samples were collected on June 13 and 14, 2011 onboard the R/V John Le Conte 867
in Lake Tahoe, CA. Two stations were sampled, West-Lake Tahoe (WLT, a.k.a. Index 868
Station, 39 05.840 N 120 09.300 W) and Mid-Lake Tahoe (MLT, 39 08.50 N 120 00.925 869
W, Figure 1). The oligotrophic lake has a surface area of 500 km
2
, maximum depth of 870
over 500 m, and an average depth of 300 m (Goldman, 1988) making it the second 871
deepest lake in the United States. It has shifted from nitrogen (N) toward phosphorus (P) 872
limitation as N inputs from anthropogenic sources have increased without a concurrent 873
increase in P (Goldman, 1988 and 2000). Lake Tahoe has a unique population of N
2
874
fixing epilithic periphyton that are active throughout the year (Reuter et al., 1986). Thus, 875
making this lake ideal for the study of limitations of both C and N
2
fixation and 876
biogeochemical cycling. 877
42
878
Figure
2-‐1.
Sampling
locations
in
Lake
Tahoe
located
on
the
California
and
Nevada
border.
879
The
two
stations,
Mid
Lake
Tahoe
(white
circle
next
to
white
state
border)
and
West
Lake
880
Tahoe
are
marked
(white
circle
on
the
west
side
of
the
lake,
Photo
credit-‐
terc.ucdavis.edu,
881
State
of
the
Lake
Report,
2012). 882
Sample Collection and Storage 883
Water samples were collected from 11 depths ranging from 10 m to 130 m at the 884
WLT station and 140 m at the MLT station using a Niskin Bottle Rosette sampler and 885
trace metal clean techniques as described by Bruland et al. (1979). A Seabird CTD was 886
utilized to collect ancillary data including temperature (° C) and salinity (PSU). 887
B-vitamin and trace metal samples were filtered with a 0.2 micron cartridge filter. 888
Vitamin samples were stored in methanol cleaned amber HDPE bottles and frozen until 889
analysis. Trace metal samples were stored in HDPE trace metal cleaned bottles at room 890
temperature until analysis. B-vitamin samples were extracted and pre-concentrated 891
according to the method of Sañudo-Wilhelmy et al. (2012). Briefly, water samples (2 L) 892
were passed through solid-phase C18 resin at a flow rate of 1 ml min
-1
to concentrate the 893
43
B-vitamins. Sample pH was adjusted to pH 6.5 before being passed through the resin, 894
and subsequently adjusted to pH 2.0 and passed through the column again to obtain 895
maximum recovery. Vitamins were subsequently eluted off the columns with methanol, 896
dried, and dissolved in 200 µl of MilliQ water. B-vitamin concentrations were then 897
quantified using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Each 898
extraction included a blank and positive control to test for contamination and extraction 899
efficiency. The limit of detection (LOD) for the technique can be as low as 0.81 pM for 900
B
1
, 0.67 for B
2
, 0.61 pM for B
6
, 0.23 for B
7
, 0.18 for B
12
, and 0.17 pM for methionine 901
(met, Sañudo-Wilhelmy et al., 2012). Samples were divided into three sets in order to 902
concentrate all samples; set 1 included all WLT station depths except 20 and 30 m, and 903
MLT station depth 20 m, Set 2 included all MLT station depths except 20, 70, and 100 m 904
and WLT station depths 20 and 30 m, and Set 3 included MLT station depths of 70 and 905
100 m. 906
Trace metals were analyzed by inductively coupled plasma mass spectrometry 907
(ICP-MS, Sañudo-Wilhelmy et al., 2001; Sañudo-Wilhelmy et al., 2004; Tovar-Sanchez 908
et al., 2006). This was preceded by acidification using 6 N optimum grade HCL (pH < 2) 909
for no less than 1 month (Sañudo-Wilhelmy and Flegal, 1996). Total dissolved water 910
column concentrations of Co, Cu, Fe, Ni, and V (bioactive trace metals), as well as Al, 911
Mn, Ti, Ba, pB, and Cd, (terrigenous metals) were quantified. The chemical speciation of 912
molybdenum (Mo(V) and Mo(VI)) were determined according to the method of Wang et 913
al. (2009). The LOD for Mo and Mo(V) was 2.8 pmol L
-1
, and the ICP-MS LOD ranged 914
from a low of 0.8 pmol L
-1
for Cd up to 250 pmol L
-1
for Ti (all representative elemental 915
44
concentrations were a minimum of an order of magnitude higher than their LODs’). 916
Subsamples of 1 L from each depth were reserved for chlorophyll a (Chl a) 917
analysis. Samples were immediately filtered onto 25 mm GF/F filters and frozen. Filters 918
were then analyzed according to the EPA modified fluorometric method 445.0 (Arar and 919
Collins, 1997) in a Turner Designs Trilogy Fluorometer within one week. Subsamples of 920
40 ml were also collected from each depth for bacteria abundance; samples were 921
preserved with 0.8 ml formalin (0.2 µm filtered, 2% vol/vol final concentration) and 922
stored at 20° C until analysis. Samples were enumerated with in two months of 923
collection. Duplicate filters were made for each sample, 5 ml sample was filtered down 924
to 1 ml before adding 50 µl DAPI stain to the sample. After 7 minutes, the sample was 925
filtered and at least 10 fields were counted with magnification of 100x using 926
epifluorescence microscopy (Hoff, 1993). 927
Results 928
Concentrations of B-Vitamins 929
Depth distributions of B-vitamins in Lake Tahoe varied spatially throughout the 930
water column and by distance from shore at the WLT and MLT stations (Figure 2). 931
Vitamin B
1
concentrations were below detection limit (BDL) at all depths at except 60 932
and 70 m (2.63 and 1.16 pM, respectively) at the WLT and was BDL at the MLT station 933
at all depths, B
2
was BDL at both stations at all depths (data not shown). Vitamin B
6
934
concentrations ranged from BDL to 3.17 pM and BDL to 3.67 pM and vitamin B
7
ranged 935
from 0.59 to 4.28 pM and 0.23 to 3.45 pM at the WLT and MLT stations, respectively 936
(Figures 2 and 3). Vitamin B
12
was BDL at all depths at the WLT station and was BDL 937
45
at all depths except 70 m (0.32 pM) at the MLT station. The amino acid met ranged from 938
BDL to 12.8 pM and BDL to 10.81 pM, respectively (Figure 2). Extraction efficiency 939
during this study was above 90% for all B-vitamins with the exception of vitamin B
12
and 940
met. Vitamin B
12
had low extraction efficiency during the WLT sample run, while met 941
had low extraction efficiency during the MLT sample run (data not shown). Percent 942
recoveries were unavailable due to variations in pH of the MQ water used to re-suspend 943
the B-vitamin samples before quantification. 944
945
46
A
946
947
B
948
949
Figure
2-‐2.
Profiles
of
dissolved
B-‐vitamin
concentrations
(pM
±
1
standard
deviation):
950
pyridoxine
(B6),
biotin
(B7),
and
cobalamin
(B12),
and
the
amino
acid
methionine
in
the
water
951
column
at
the
(A)
West
Lake
Tahoe
station
and
(B)
Mid
Lake
Tahoe
stations.
952
953
47
A
954
955
B
956
957
Figure
2-‐3.
Profiles
of
dissolved
(A)
pyridoxine
and
(B)
biotin
concentrations
(pM,
average
±
1
958
standard
deviation)
in
the
water
column
at
the
West
and
Mid
Lake
Tahoe
stations
(WLT
and
959
MLT,
respectively).
960
Trace Metals 961
Trace metal concentrations were all in the nM range, and varied spatially (Figure 962
4). In general, the WLT station had higher trace metal concentrations compared to the 963
48
MLT station. For many of the metals at WLT there were peaks observed at or near 10 964
and 50 m. The MLT station peaks were more variable with depth and were much lower 965
in magnitude compared to the WLT station. The ranges for WLT and MLT stations for 966
most trace metals were less than 100 nM except for aluminum and zinc that were found 967
to be in the less than 300 nM range (Table 1). 968
49
969
Figure
2-‐4.
(A)
Profiles
of
dissolved
trace
metal
concentrations
(nM)
in
the
water
column
at
970
the
West
and
Mid
Lake
Tahoe
stations
(WLT
and
MLT,
respectively).
971
50
972
Figure
2-‐4.
(B)
Continuation:
profiles
of
dissolved
trace
metal
concentrations
(nM)
in
the
973
water
column
at
the
West
and
Mid
Lake
Tahoe
stations
(WLT
and
MLT,
respectively). 974
Biological parameters
975
Chl a concentrations at WLT and MLT stations averaged 1.29 and 0.99 µg L
-1
976
(range: 0.51 to 2.38 µg L
-1
and 0.14 to 2.31 µg L
-1
, respectively, Figure 5). The Chl a 977
maximum was located at a depth of 50 m at the WLT station and 40 m at the MLT 978
station. Bacterial abundance at WLT and MLT stations averaged 4.1 x 10
5
and 4.2 x10
5
979
cells ml
-1
(range: 3.4 x 10
5
to 5.1 x 10
5
cells ml
-1
and 2.6 x 10
5
to 5.9 x 10
5
cells ml
-1
, 980
respectively, Figure 5). The bacterial profile at WLT showed a primary peak at 50 m 981
with a smaller secondary peak at 20 m as well as increased cell abundance from 90 to 130 982
51
m. The MLT bacterial profile also had a primary peak located at 50 m and increased cell 983
abundance from 100 to 140 m. No secondary peak was observed at the MLT station. 984
985
Figure
2-‐5.
Depth
profiles
of
(A)
Chlorophyll
a
(Chl
a)
concentrations
(mg
L
-‐1
,
average
±
1
986
standard
deviation)
and
(B)
Bacterial
abundance
(cells
ml
-‐1
,
average
±
1
standard
deviation)
987
at
the
West
and
Mid
Lake
Tahoe
stations
(WLT
and
MLT,
respectively). 988
989
52
Water Column Characteristics 990
Temperature at the WLT station ranged from 8.2 C at 10 m to 5.3 C at 130 m and 991
from 7.6 C at 10 m to 5.3 C at 140 m at the MLT station. Salinity at the WLT and MLT 992
stations ranged from 0.044 at 10 m to 0.42 at 130 m, and from 0.043 to 0.42 at 140 m, 993
respectively (Figure 6). 994
995
Figure
2-‐6.
Ancillary
data:
temperature
(C)
and
salinity
(PSU).
Data
collected
from
a
CTD
996
sensor
at
the
West
and
Mid
Lake
Tahoe
stations
(WLT
and
MLT,
respectively).
997
Discussion 998
Measuring B-vitamins in freshwater systems, such as Lake Tahoe, continues to be 999
difficult. The results of this study were similar to previous studies showing most B- 1000
vitamins were BDL. Vitamin B
1
was BDL at all depths except for 2 at the WLT station 1001
and B
2
was BDL at both stations at all depths. Vitamin B
12
was BDL at all depths at the 1002
WLT station and at the MLT station, except at 70 m. The amino acid met was BDL at 1003
most depths except for 10, 40 and 50 m at the WLT station and 80 m at the MLT station. 1004
Vitamin B
6
was generally above the limit of detection at the WLT station except at 1005
53
depths of 20 and 30 m. However, vitamin B
6
concentrations at the MLT station were 1006
mostly BDL except for 20, 40, 70, and 100 m depths (Figure 2). These results suggest 1007
that most B-vitamin concentrations in Lake Tahoe were very low during this study, which 1008
is consistent with previous results (Carlucci and Bowes, 1972). It can be concluded that 1009
sample volumes of 2 L were insufficient to raise the concentration above the limit of 1010
detection (LOD) for most B-vitamins in Lake Tahoe. Therefore, increasing sample 1011
volumes to at least 10 L is suggested for future studies of the direct determination of B- 1012
vitamins from oligotrophic freshwater systems. A technical error occurred during sample 1013
analysis, in which the pH of the water used to re-suspend the concentrated vitamins 1014
before injection into the LC/MS/MS varied and altered the recoveries of some B-vitamins 1015
(Sañudo-Wilhelmy, pers. comm.). The effect was minimal for vitamins B
2
, B
6
, and B
7
. 1016
However, it had a larger effect on the remaining vitamins (Sañudo-Wilhelmy, pers. 1017
comm., data not shown). Therefore, the discussion will focus on vitamins B
6
and B
7
as 1018
they were both above the LOD and minimally affected by the variation in pH. 1019
Vitamin-B
6
concentrations at the WLT station decreased with depth except 1020
between 20 and 30 m where concentrations were BDL (Figure 3). The highest values 1021
were found at depths of 10 (3.17 ± 0.19 pM) and 40 m (2.953 ± 0.28). This suggests that 1022
potential sources of this vitamin include external sources as well as production in the 1023
surface water by microorganisms. Variability with depth was also observed at the MLT 1024
station with the highest concentrations measured at 20 m (3.69 ± 0.25) and 100 m (2.22 ± 1025
0.35) depths. Smaller concentrations were measured at 40 m (1.21 ± 0.26) and 70 m 1026
(1.14 ± 0.17) depths, and concentrations were BDL at the other depths (Figure 3). This 1027
54
suggests that there is the potential for vitamin B
6
to limit or co-limit biological 1028
productivity in this region of Lake Tahoe and that this vitamin is likely produced by 1029
organisms in the water column. Concentrations were lower than the only other known 1030
study to directly measure vitamin B
6
in a freshwater system, Lake Michigan, which 1031
ranged from undetectable to 1500 pM (Sañudo-Wilhelmy, pers. comm.). Further studies 1032
are needed to identify sources, sinks, and to determine the influence of vitamin B
6
on 1033
biological productivity in different regions of Lake Tahoe and other freshwater systems. 1034
Vitamin B
7
concentrations varied with depth at the WLT station, large peaks were 1035
measured at depths of 10 m (3.2 pM ± 1.3) and between 40 to 50 m (averaging 3.7 pM ± 1036
0.7). At depths from 60 to 130 m, vitamin B
7
concentrations were lower and much less 1037
variable (averaging 1.5 pM ± 0.22). The lowest concentrations were found between 20 1038
and 30 m (averaging 0.6 pM ± 0.04, Figure 3). MLT vitamin B
7
concentrations were also 1039
variable with depth with peaks measured at 20, 70, and 100 m (3.4 ± 0.4, 1.2 ± 1.1, and 1040
2.3 ± 0.2 pM, respectively, Figure 3). The previous studies conducted near the center of 1041
Lake Tahoe found that vitamin B
7
was detectable in August of 1969 at depths from only 1042
25 to 60 m ranging from 6.0 and 1.7 ng L
-1
. However, it was undetectable throughout the 1043
water column (1-400 m) in February of 1970 (Carlucci and Bowes, 1972). Previous 1044
studies have shown that microorganisms in sediments can be responsible for the 1045
production of B-vitamins (Provasoli, 1958; Burkholder 1963). Slight increases were 1046
observed with depth (Figure 3) suggesting that the sediments are a possible source of 1047
vitamins to the water column. Further studies investigating the microorganisms present 1048
55
and vitamin production rates are required to determine the influence of sediments to 1049
water column B-vitamin concentrations in Lake Tahoe. 1050
Vitamin B
7
concentrations measured around 10 m µg L
-1
in Lake Sagami, Japan 1051
during the spring circulation (March 9, 1970) when increased winds result in deeper 1052
vertical water column mixing. However, during the summer stratification event (June 8, 1053
1970), surface concentrations were as high as 40 m µg L
-1
before decreasing rapidly near 1054
the thermocline. An increased concentration with depth was mainly observed during the 1055
summer stratification period. However, a slight increase was observed during the spring 1056
circulation (Ohwada and Taga, 1972). Seasonal cycles showed similar patterns with the 1057
highest concentrations occurring during the summer months along with the sediments 1058
providing a source of vitamins to the deep water (Ohwada and Taga, 1972). This 1059
suggests that higher concentrations may also be present in Lake Tahoe during the 1060
summer stratification events. We suggest increased temporal sampling to determine the 1061
possible effects of thermocline formation on vitamin concentrations. 1062
Depth profiles of vitamins B
6
and B
7
from Lake Michigan ranged from 1063
undetectable to 1500 and 40 pM, respectively (Sañudo-Wilhelmy, pers. comm.). Vitamin 1064
distributions were similar to those from Lake Tahoe being highly variable with depth and 1065
often completely absent or below the limit of detection. Specifically, vitamin B
6
was 1066
BDL at all depths from one station and B
7
was BDL at another station between 50 and 1067
100 m. This suggests that organic growth factors such as B-vitamins can limit or co-limit 1068
biological processes in freshwater lakes across the continental U.S. 1069
56
Trace metals are potential factors affecting the production and consequently the 1070
concentrations of B-vitamins in freshwater systems. Vitamin B
7
for instance, requires the 1071
trace metals Fe and S for vitamin synthesis (Berkovitch et al., 2004). Proteins containing 1072
an iron-sulfur cluster have been shown to be required for proper functioning of biotin 1073
synthase (Mühlenhoff et al., 2007). Trace metal concentrations in Lake Tahoe were 1074
below 300 nM and most concentrations were similar to those measured in 2009 at both 1075
the WLT and MLT stations (Table 1, Romero et al., 2013). The trace metals Al, Ti, and 1076
Fe were higher in 2011 compared to those measured in 2009 at the WLT and MLT 1077
stations, as well as Cu at the WLT station. Higher concentrations of trace metals were 1078
measured in the Truckee River that empties into Lake Tahoe (Benson, 1984; Johannesson 1079
et al., 1997). This suggests that rivers are a potential source of trace metals to Lake 1080
Tahoe, and that B
7
production or growth of B
7
synthesizing organism at the MLT station 1081
could be limited by Fe availability as Fe concentrations were lower then those at the 1082
WLT station (Figure 4). Specific trace metal concentrations vary from one another 1083
emphasizing the importance of measuring each one individually. They also vary by 1084
geographic location (Table 1) and could therefore play various roles in the different 1085
ecosystems. This suggests that further studies in different freshwater lakes can increase 1086
our understanding of the effects of specific trace metals on B-vitamin production and 1087
availability.
1088
1089
57
Table
2-‐1.
Trace
metal
concentrations
at
various
freshwater
lakes
and
rivers
(WLT
1090
and
MLT,
West
and
Mid
Lake
Tahoe
stations,
respectively). 1091
1092
Similar patterns in the WLT station depth profiles of vitamin B
6
and different 1093
trace metals were observed (Figure 7). Vitamin B
6
and certain trace metal (specifically 1094
Pb, Ni, Cu, and Zn) concentrations tended to increase together (Table 2). Similar patterns 1095
in the WLT station depth profiles of vitamin B
7
and trace metals (particularly Ag, Cd, Pb, 1096
Al, Mn, Ni, Cu, and possible Fe) were observed with peaks near the surface at 10 m and 1097
at 50 m depths (Figure 7). Vitamin B
7
and certain trace metals (specifically Ag, Pb, Al, 1098
Fe, and Ni) tended to increase together (Table 2). The corresponding pattern between Fe 1099
and vitamin B
7
may be indicative of Fe limitation of vitamin B
7
biosynthesis. However, 1100
further studies investigating the stimulation of vitamin B
7
production with varying 1101
concentrations of Fe are suggested. Similar patterns were not observed at the MLT 1102
58
station between vitamins B
6
and B
7
, and trace metal concentrations (Figure 8). No 1103
significant relationships at the MLT station were observed between the two B-vitamins 1104
and any of the measured trace metals (Table 2). This suggests that trace metals may be 1105
playing less of a role limiting B-vitamin concentrations at the MLT station. 1106
59
1107
Figure
2-‐7.
(A)
Profiles
of
dissolved
pyridoxine
and
biotin
(pM,
average
±
1
standard
1108
deviation)
with
trace
metal
concentrations
(nM)
in
the
water
column
at
the
West
Lake
Tahoe
1109
station.
1110
60
1111
Figure
2-‐7.
(B)
Continuation:
profiles
of
dissolved
pyridoxine
and
biotin
(pM,
average
±
1
1112
standard
deviation)
with
trace
metal
concentrations
(nM)
in
the
water
column
at
the
West
1113
Lake
Tahoe
station.
1114
61
1115
Figure
2-‐8.
(A)
Profiles
of
dissolved
pyridoxine
and
biotin
(pM,
average
±
1
standard
1116
deviation)
and
trace
metal
concentrations
(nM)
in
the
water
column
at
the
Mid
Lake
Tahoe
1117
station. 1118
62
1119
1120
Figure
2-‐8.
(B)
Continuation:
profiles
of
dissolved
pyridoxine
and
biotin
(pM,
average
1121
±
std)
and
trace
metal
concentrations
(nM)
in
the
water
column
at
the
Mid
Lake
Tahoe
station. 1122
The source of trace metals at the WLT station is consistent with input from the 1123
intrusion of groundwater from the surrounding environment. In fact, the Ward Valley 1124
aquifer located near the WLT station reaches a depth of 50 m and was shown to be a 1125
significant source of both N and P to Lake Tahoe (Goldman, 1979; Nagy, 2003). This 1126
suggests that it may also be a source of trace metals to Lake Tahoe. A common indicator 1127
of groundwater intrusion, a decrease in water temperature, was not observed during this 1128
63
study at 50 m (Figure 6). Thus, further studies are needed to confirm the sources of trace 1129
metals to the WLT station. Depth profiles of trace metals at the MLT station showed less 1130
variation (Figure 4) and were likely less influenced by the possible intrusion of 1131
groundwater. Higher trace metal surface concentrations suggest wind deposition as a 1132
possible source. However, further studies are required to determine the different sources 1133
of trace metals to the MLT station. 1134
1135
64
Table
2-‐2.
Pearson
product
moment
correlation
analysis
of
vitamins
B6
and
B7,
and
trace
1136
metals
at
the
West
and
Mid
Lake
Tahoe
stations
(WLT
and
MLT,
respectively).
R
=
correlation
1137
coefficient
1138
1139
Chl a concentrations at the WLT and MLT stations ranged from 0.5 to 2.5 and 2.0 1140
µg L
-1
, respectively (Figure 5). They were similar to those measured in October 2009 at 1141
WLT and MLT at 50 m (2.0 µg L
-1
± 0.1 and 1.3 µg L
-1
± 0.2, respectively) and 70 m (1 1142
65
µg L
-1
± 0.1 and 2.2 µg L
-1
± 0.2, respectively, Romero et al., 2013). These values were 1143
also consistent with depth profiles that showed the deep Chl a maximum could be as high 1144
as 6 µg L
-1
, however, values closer to those measured in this study were also observed 1145
(Alumbaugh et al., 2012). Large variations in Chl a concentrations have been previously 1146
observed, however they were generally lower than measured values during this study. 1147
For instance, summer surface values ranged from 0.11 to 0.22 mg m
-3
but at a depth of 1148
100 m it ranged from 0.59 to 0.88 mg m
-3
(Coon et al., 1987). Another study found water 1149
column values ranged from 0.15 to 0.50 mg m
-3
and daily transects in different directions 1150
resulted in significantly different variance spectra (Abbott et al., 1982). This suggests 1151
that phytoplankton dynamics are variable in Lake Tahoe, and further studies are needed 1152
to determine the influence of B-vitamins on phytoplankton biomass and production. 1153
Bacterial abundance varied between stations and with depth (Figure 5). Both 1154
depth profiles showed peaks at 50 m, however higher abundances were observed at the 1155
MLT station. A peak in B
7
concentration (Figure 3) corresponded to the highest Chl a 1156
and bacterial abundance at the WLT station (Figure 5). Local production of vitamin B
7
1157
could be due to phytoplankton production; however, it was just as likely produced by the 1158
bacterial community as both populations increased. Previous studies have found the 1159
production of B-vitamins by planktonic bacteria in Lake Jeziorak (Donderski and Sokol, 1160
1990) and Lake Jasne (Donderski and Nowacka, 1992). Slight increases at the deepest 1161
depths in the profiles were also observed in vitamin B
7
concentrations and bacterial 1162
abundance (Figures 3 and 5), suggesting that it may have originated from the bacterial 1163
population. Further studies are required to determine the magnitude of the contribution 1164
66
of different sources to dissolved vitamin B
7
concentrations. Determination of the sources 1165
and sinks in future studies would help shed light into the potential cycling of B-vitamins 1166
through different aquatic communities and environments. 1167
C and N
2
fixation rates were determined at depths of 0, 50 and 80 m at the WLT 1168
station and 50 m at the MLT station (Figure 9, Gunderson pers. comm.). Productivity at 1169
50 m at MLT (0.005 ± 0.0003 nmol L
-1
hr
-1
) was less than that at WLT (0.1 ± 0.03 nmol 1170
L
-1
hr
-1
). This study could not determine the role that vitamin B
7
plays in C fixation. 1171
However, higher rates of C fixation were observed with higher concentrations of vitamin 1172
B
7
(Figures 3 and 9). This suggests that primary producers and thus C fixation may be 1173
limited or co-limited by vitamin B
7
. N
2
fixation was observed at the WLT station only 1174
with similar values at the surface and 50 m (0.15 nmol ± 0.13 L
-1
hr
-1
and 0.2 ± 0.3 nmol 1175
L
-1
hr
-1
, respectively), but decreased at 80 m to 0.05 ± 0.04 nmol L
-1
hr
-1
). Higher 1176
vitamin B
6
concentrations were observed at 10 and 50 m depths compared to 80 m 1177
(Figures 3 and 9). However, with the limited data it is difficult to conclude if vitamin B
6
1178
acts as a limiting nutrient to N
2
fixation in Lake Tahoe. Future studies are needed to 1179
determine the role B-vitamins play in C and N cycling in Lake Tahoe. 1180
67
1181
Figure
2-‐9.
di-‐nitrogen
(N2)
and
carbon
(C)
fixation
(nmol
L
-‐1
hr
-‐1
,
average
±
1
standard
1182
deviation)
at
the
West
and
Mid
Lake
Tahoe
stations
(WLT
and
MLT,
respectively). 1183
Vitamin B
6
and B
7
concentrations in the oceans were found to be spatially 1184
variable, and reach maximums in the hundreds of pM range (Sañudo-Wilhelmy et al., 1185
2012). Consequently, B-vitamin limitations may be more predominant in FW systems 1186
with concentrations in the 0-5 pM range. However, with the wide range of values 1187
observed in the oceans and the vast areas observed with no measurable values, variation 1188
may be a function of differences in sample size from each system. Therefore, we suggest 1189
that further studies increase sampling frequency temporally and spatially along with 1190
sample volume. We were unable to calculate percent recoveries and pH variations in the 1191
water used to re-suspended samples which also contributed to the possibility of errors. 1192
We also suggest that future studies would benefit from increased controls, replications, 1193
and standards. Our understanding of the ecological significance of B-vitamins to global 1194
biogeochemical cycles will continue to increase as we gain knowledge of B-vitamins 1195
from various freshwater systems, such as Lake Tahoe. 1196
68
Acknowledgments 1197
We thank A. Liston, Lake Tahoe boat captains, and support staff (Tahoe Environmental 1198
Research Center) for their support in the field and laboratory. We also thank the 1199
following people for their specific contributions and for providing both field and 1200
laboratory assistance: A. M. Liss, M. Tiahlo, trace metal data was collected and analyzed 1201
by N. J. Klein, B-vitamins were analyzed by L. Cutter, and isotopic nitrogen and carbon 1202
fixation data were collected and analyzed by T. Gunderson. 1203
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73
CHAPTER THREE: SUMMARY 1340
B-vitamins function as co-enzymes and catalysts for many of the most basic 1341
metabolic reactions. This suggests these factors will influence aquatic species 1342
composition, abundance, and rates of productivity. The carbon (C) and nitrogen (N) 1343
biogeochemical cycles are linked by the inherent chemical requirements of living 1344
organisms due to the composition of biological molecules, and both are important 1345
regulators of global processes. Thus, the regulation of enzymatic reactions that are 1346
dependent on the availability of organic growth factors are one mechanism driving global 1347
biogeochemical cycling. This dissertation investigates the abundances, distributions, and 1348
influences on biogeochemical cycling of some of the most important environmental 1349
organic growth factors, vitamins B
1
, B
6
, and B
7
, in two contrasting aquatic systems. 1350
The Western Tropical North Atlantic (WTNA) Amazon River plume is 1351
characterized as an eutrophic marine system that has high rates of productivity (i.e. 1352
elevated rates of di-nitrogen (N
2
) and C fixation) which support increased C export. This 1353
study covered a vast area in the WTNA, with highly variable sea surface salinities 1354
ranging from almost freshwater to typical oceanic salinity. All biogeochemical processes 1355
investigated in this study were taking place in the water column. This is contrasted with 1356
Lake Tahoe, C.A., which is characterized as an oligotrophic high alpine freshwater 1357
system. Lake Tahoe is known to have a benthic community of N
2
fixing organisms that 1358
is active year round with lower rates of C fixation than is found in the WTNA Ocean. 1359
Vitamins B
1
and B
6
in the WTNA Amazon River plume, and vitamins B
6
and B
7
in Lake 1360
Tahoe were measured and found to be above the limit of detection. Vitamins B
1
and B
7
1361
74
are involved in reactions involving C transformations, and can therefore influence the C 1362
biogeochemical cycle. While vitamin B
6
is involved in many reactions involving amino 1363
acids transformation and can therefore influence the N biogeochemical cycle. Thus, 1364
these B-vitamins will have various influences on global biogeochemical cycling. 1365
The first direct measurements of vitamin B
1
and B
6
in the WTNA Amazon River 1366
plume ranged from below the limit of detection to 230 and 40 pM, respectively. There 1367
was a significant influence of the Amazon River plume on vitamin B
1
concentrations, 1368
with higher concentrations measured in the lower salinity plume water compared to 1369
typical oceanic salinity water below the plume. However, vitamin B
6
concentrations in 1370
the river plume water were not significantly different than those measured below the 1371
plume. This suggests that the Amazon River may be a source of some B-vitamins to the 1372
WTNA Ocean. The vitamins B
1
and B
6
were also shown to affect rates of C and N
2
1373
fixation in this region. Significantly higher rates of C and N
2
fixation were observed 1374
with higher vitamin B
1
concentrations at low and intermediate (mesohaline) stations. 1375
Significantly higher rates of N
2
fixation co-occurred with higher concentrations of 1376
vitamin B
1
at a mesohaline station and with higher concentrations of vitamin B
6
at a low 1377
salinity station, both without concurrent elevated rates of C fixation. The results of this 1378
work suggest that there may be a relationship between B-vitamin concentrations and 1379
elevated rates of C and N
2
fixation, which may directly affect biogeochemical cycles in a 1380
vast and highly productive area of the WTNA Ocean. This emphasizes the importance of 1381
B-vitamins on marine systems, especially the influence they have on rates of 1382
biogeochemical cycles, ecosystem structure, and function. 1383
75
The first direct measurements of vitamin B
6
and B
7
concentrations were 1384
undertaken in Lake Tahoe, CA and ranged from below the limit of detection to 4 pM and 1385
from 0.2 to 4.5 pM, respectively. The other B-vitamins were generally undetectable 1386
suggesting that larger sample volumes (at least 4 L) are required for this assay in 1387
freshwater systems. Further studies will be required to determine if the other B-vitamins 1388
are present in freshwater at high enough concentrations to be directly measured. In fact, 1389
vitamin B
6
concentrations in Lake Tahoe were found to be 10 times lower then those 1390
measured in the WTNA. This suggests that dissolved B-vitamins in the water column in 1391
freshwater systems may be limiting or co-limiting metabolic processes. Further research 1392
investigating vitamin requirements and uptake kinetics will provide valuable information 1393
on the significance of exogenous supplies of the specific B-vitamins in different aquatic 1394
habitats. 1395
The potential relationship between B-vitamins and trace metal concentrations was 1396
also investigated in Lake Tahoe. In general, trace metal concentrations were higher at the 1397
WLT station compared to the MLT station and found in the nM ranges. This suggests a 1398
terrestrial source of trace metals to Lake Tahoe as higher concentrations were measured 1399
at the near shore station. No significant relationships were observed between trace 1400
metals and B-vitamin concentrations at the MLT station and less variation was measured 1401
through the water column. However, correlation analysis results showed that 1402
concentrations of some trace metals and vitamins B
6
and B
7
tended to positively correlate 1403
at the WLT station. Specifically, Pb, Ni, Cu, and Zn increased with vitamin B
6
, and Ag, 1404
Cd, Pb, Al, Mn, Ni, Cu, and Fe increased with vitamin B
7
at the WLT station. Peaks near 1405
76
the surface at 10 and 50 m depths were also observed with these trace metals and vitamin 1406
B
7
concentrations. These patterns suggested the potential for trace metal and B-vitamin 1407
co-limitation in Lake Tahoe. 1408
Biological measures of chl a concentrations ranged from 0.5 to 2.5 and 2.0 µg L
-1
1409
and bacterial cell counts ranged from 3.4 x 10
5
to 5.1 x 10
5
cells ml
-1
and 2.6 x 10
5
to 5.9 1410
x 10
5
cells ml
-1
at the WLT and MLT stations, respectively. Similar patterns with peaks 1411
in vitamin B
7
and these biological parameters suggest that they may be important sources 1412
to the water column. Similar patterns of vitamin B
6
concentrations and these biological 1413
parameters were not observed during this study. Previous studies have found B-vitamin 1414
production by phytoplankton and planktonic bacterial populations from freshwater 1415
systems. This suggests that the different B-vitamins behave independently and highlights 1416
the importance of detailed studies investigating each one separately using appropriate 1417
individualized methods. Direct studies are required to determine the specific sources and 1418
sinks of the various B-vitamins in Lake Tahoe. 1419
Rates of biological C and N
2
fixation were measured at three corresponding 1420
depths during this study. C fixation was higher at the WLT station at 50 m and 1421
corresponded to higher concentrations of vitamin B
7
. Concentrations of vitamin B
6
were 1422
similar at both stations at a depth of 50 m. This suggests that the biological community 1423
may have been limited or co-limited by vitamin B
7
concentrations in the water column. 1424
N
2
fixation at the WLT station was higher at the surface and at 50 m compared to 80 m 1425
and similar patterns were observed in vitamin B
6
and B
7
concentrations. This suggests 1426
that there is the potential for B-vitamin limitation of biologically mediated C and N
2
1427
77
fixation in Lake Tahoe as well as the WTNA Ocean. However, additional research will 1428
be needed to determine the magnitude and importance of specific effects of B-vitamins 1429
on biogeochemical cycling in the various aquatic systems. 1430
This dissertation highlights the importance of B-vitamins as growth factors that 1431
influence aquatic community structure, composition, and function. With the application 1432
of new methodology we are just beginning to be able to explore different aquatic systems 1433
as environmental concentrations were often below the limit of detection using the older 1434
methodology. This is allowing researchers to provide the necessary baseline 1435
measurements in order to study the effects of B-vitamins on important global processes 1436
such as C and N
2
fixation. B-vitamin auxotrophy has been found to be widespread 1437
among plankton species and thus exogenous supplies have the potential to be vital 1438
regulators of biogeochemical cycling. In fact, significant increases in C and N
2
fixation 1439
and higher B-vitamin concentrations were observed in the highly productive WTNA 1440
Ocean due to the Amazon River influx. Further studies are needed to determine the 1441
magnitude and influence they have in freshwater systems such as Lake Tahoe. However, 1442
some B-vitamin concentrations in both systems were influenced by proximity to shore 1443
and suggest freshwater intrusion from land (the Amazon river in the WTNA and 1444
groundwater in Lake Tahoe) may be an important source of B-vitamins to aquatic 1445
systems. These studies show the dynamic interactions between B-vitamins and key 1446
biogeochemical cycling emphasizing the potential they have to alter community structure 1447
that results in changes in ecosystem function and rates of global biogeochemical cycling. 1448
78
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limitation of nitrogen fixation by Trichodesmium in the central Atlantic Ocean. 1739
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Sañudo-Wilhelmy, S.A., Tovar-Sanchez, A., Fu, F., Capone, D. G., Carpenter, E. J., 1741
Hutchins, D. A., (2004). The impact of surface-adsorbed phosphorus on 1742
phytoplankton Redfield stoichiometry. Nature 432, 897-901. 1743
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Sañudo-Wilhelmy, S.A. (2006). Oceanography: A phosphate alternative. Nature 439, 25- 1744
26. 1745
Sañudo-Wilhelmy, S. A., Lynda, C., Durazo, R., Smail, E., Gomez-Consarnau, L., Webb, 1746
E. A., Prokopenko, M., Karl, D. M., and Berelson, W. M. (2012). Multiple B- 1747
vitamin deficiency in large areas of the coastal ocean. Proc. Natl. Acad. Sci. 109, 1748
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Snell, E. E. (1953). Metabolic significance of B-vitamins. Physiol. Rev. 33, 509–524. 1751
Sohm, J. A., Subramaniam, A., Gunderson, T. E., Carpenter, E. J., and Capone, D. G. 1752
(2011a). Nitrogen fixation by Trichodesmium spp. and unicellular diazotrophs in 1753
the North Pacific Subtropical Gyre. J. Geophys. Res. 116, 2156-2202. 1754
Sohm, J.A., Capone, D. G. (2006). Phosphorus dynamics of the tropical and subtropical 1755
north Atlantic: Trichodesmium spp. versus bulk plankton. Mar. Ecol. Prog. Ser. 1756
317, 21-28. 1757
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limitation of Trichodesmium spp. in the North Pacific, North Atlantic, and the 1759
North Coast of Australia. Limnol. Oceangr. 53, 2495-2502 1760
Sohm, J. A., Webb, E. A., and Capone, D. G. (2011b). Emerging patterns of marine 1761
nitrogen fixation. Nat. Rev. Microbiol. 9, 499–508. 1762
Staley, J. T., Gunsalus, R. P., Lory, S., Perry, J. J., (2007). Microbial Life, 2
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Strickland, J. D. H. (2009). Vitamin B12, thiamine, biotin. The ecology of the 1765
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A., Agustí,
S.,
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A.,
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G.,
Duarte,
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M. (2006). Effects of dust deposition and 1776
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and Subtropical North Atlantic Ocean. Limnol. Oceanogr. 51, 1755-1761. 1778
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on dissolved and intra-cellular metal concentrations in Trichodesmium colonies 1780
along the western boundary of the sub-tropical North Atlantic Ocean. 1781
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North Atlantic Ocean. Science 289, 759–762. 1803
89
APPENDIX: PHOSPHONATE UTILIZATION AND METHANE PRODUCTION IN 1804
FIELD POPULATIONS OF TRICHODESMIUM SPP. FROM THE WESTERN 1805
TROPICAL NORTH ATLANTIC OCEAN AND CULTURES OF 1806
TRICHODESMIUM ERYTHRAEUM IMS101 1807
Abstract 1808
Phosphonates are organic compounds containing at least one carbon-phosphorus 1809
(C-P) bond, and are present in oligotrophic oceans. They were previously thought to be 1810
unavailable to Trichodesmium, a prominent marine cyanobacteria, until the discovery of 1811
the genes responsible for encoding proteins of the C-P lyase pathway. Trichodesmium 1812
erythraeum IMS101 and cultures containing only the associated microbial community 1813
(AMC) were both capable of growth using methylphosphonate (MnP) as the sole source 1814
of P. No statistical differences were observed in optical density at a wavelength of 600 1815
nm (OD
λ600nm
, a proxy for growth) during the stationary phase in cultures of T. 1816
erythraeum IMS101 and those containing only the AMC when grown with PO
4
3-
, MnP, 1817
or both P sources (OD
λ600nm
= 0.2, 0.3, and 0.2, respectively). However, growth was 1818
significantly higher in all P treatments compared to P deplete controls in cultures 1819
containing T. erythraeum IMS101 and the AMC (OD
λ600nm
= 0.005 and 0.008, 1820
respectively). Methane (CH
4
) accumulation was monitored daily, and showed significant 1821
increases in cultures of T. erythraeum IMS101 and the AMC when grown on MnP. 1822
Maximum CH
4
concentrations (mean ± std.) in the gas phase were measured on day 5 in 1823
the treatments containing T. erythraeum IMS101 (1.6 ± 0.05 µM) and the AMC only (1.6 1824
± 0.01 µM), but remained low in the abiotic controls (0.03 ± 0.02 µM). Low CH
4
1825
production in the treatments containing both P sources suggests that PO
4
3-
may be the 1826
90
preferred P source for cultures of T. erythraeum IMS101 and cultures containing only the 1827
AMC. Alternatively, CH
4
consumption by the AMC could be possible in the treatment 1828
containing both P sources. The lack of production in the the AMC could be explained by 1829
P limitation of the AMC in treatments containing only MnP, however further studies are 1830
required to discern specific metabolic processes of the AMC. The ecological relevance 1831
of MnP use was investigated using field populations of Trichodesmium incubated with 1832
PO
4
3-
or MnP. Similar growth patterns were observed in both P treatments and the P 1833
deplete control, with an increase on day 2 followed by a decrease. CH
4
concentrations in 1834
the MnP treatment (10.8 ± 1.0 µM) were significantly higher than in the PO
4
3-
treatment 1835
and the P deplete control (2.2 ± 0.3 and 2.1 ± 0.4 µM, respectively). This suggests the 1836
potential for methane production in the surface ocean of the WTNA associated with MnP 1837
utilization. This work will lead to a better understanding of the impact of MnP on 1838
biogeochemical cycles, CH
4
production, and the surface ocean methane paradox. 1839
Introduction 1840
Trichodesmium is a photosynthetic marine cyanobacterium that can convert 1841
atmospheric di-nitrogen (N
2
) gas to combined nitrogen (N) species, and is responsible for 1842
the majority of N
2
fixation in some areas of the tropical oceans. N
2
fixation is estimated 1843
to provide between 20 and 40 Tg of combined N species per year (Gruber and Sarmiento, 1844
1997). In the North Pacific Ocean gyre, long term studies showed that cyanobacterial N
2
1845
fixation could support up to half of the new production (Karl et al., 1997). Unlike nitrate 1846
flux from depth, which is accompanied by stoichiometric amounts of CO
2
, surface ocean 1847
N
2
fixation can promote a net uptake of CO
2
from the atmosphere. With net uptake, 1848
91
some biomass may be sequestered to the deep ocean, effectively removing atmospheric 1849
CO
2
. Atmospheric gas composition with regard to CO
2
and O
2
is largely controlled by 1850
oceanic processes such as production and consumption and contributes to variations in 1851
global temperatures (Hays et al., 2005) and surface ocean pH (Hood et al., 2004). 1852
Therefore, understanding biogeochemical cycling and gas production in the oceans will 1853
allow for a more comprehensive evaluation of how these processes might be affected as 1854
fluctuations in environmental conditions occur. 1855
Extensive studies investigating the factors that control Trichodesmium production 1856
and N
2
fixation rates show limitation varies by geographic location, and includes both 1857
physical and chemical factors such as light (Falkowski et al., 1998; Breitbarth et al., 1858
2008), temperature (Breitbarth et al., 2007), oxygen (Gallon, 1992), CO
2
(Levitan, 2007; 1859
Hutchins et al., 2007), macronutrients, and micronutrients. N
2
fixers have an increased 1860
cell quota for trace metals. In particular, iron (Fe, Raven, 1988, for a review see Sohm et 1861
al., 2011) and molybdenum (Berman-Frank et al., 2001; Sañudo-Wilhelmy et al., 2001; 1862
Kustka et al., 2003) which are required by the nitrogenase enzyme. Since trace metals in 1863
the environment are found in low concentrations, usually in the nM and pM ranges, they 1864
can often limit production and N
2
fixation rates (Morel and Price, 2003). Organic growth 1865
factors such as B-vitamins are also known to affect production as many marine 1866
planktonic species have been shown to be auxotrophic for at least one B-vitamin (Croft et 1867
al., 2006; Tang et al., 2010). However, P is thought to be the key primary limiting 1868
nutrient (Tyrrell, 1999; Wu et al., 2000), and is found in low concentrations in many 1869
oceanic regions. The ecological relevance of P has been demonstrated by studies showing 1870
92
it to be a limiting nutrient (Sañudo-Wilhelmy et al., 2001; Rees et al., 2006; Moutin et al., 1871
2008; Sohm and Capone, 2008), and more specifically, Dyhrman et al. (2002) found 1872
Trichodesmium to be P stressed in the Western North Atlantic. 1873
Biological molecules that contain P have diverse and essential cellular functions 1874
and can be found in DNA and RNA, ATP and other energy storage molecules, as well as 1875
in cellular phospholipid bilayers. The oceanic P pool consists of dissolved inorganic P 1876
(DIP) and dissolved organic P (DOP) (Benitez-Nelson, 2000). The DOP pool is 1877
comprised of nearly 75% phosphoesters and 25% phosphonates (Dyhrman et al., 2009) 1878
which are organic compounds containing at least one C-P bond. DIP is the preferred 1879
source of P as it is the most bioavailable and requires the least amount of energy for 1880
assimilation into biomass. However, marine bacteria have evolved ways to metabolize 1881
DOP (Dyhrman et al., 2007; Van Mooy et al., 2009) since DIP is often limiting (Cotner et 1882
al., 1997; Dyhrman et al., 2002). Phosphonates were previously thought to be 1883
unavailable to Trichodesmium until the discovery of the genes responsible for encoding 1884
proteins for the carbon-phosphorus (C-P) lyase pathway (Dyhrman et al., 2006; Sañudo- 1885
Wilhelmy, 2006), allowing them to utilize the P from these organic compounds. In 1886
addition, gene expression from this pathway was found in environmental populations of 1887
Trichodesmium in the Sargasso Sea (Dyhrman et al., 2006). Further, Sañudo-Wilhelmy 1888
et al. (2001) found rates of N
2
fixation to have a high positive correlation to P content of 1889
Trichodesmium colonies. The ability to utilize the DOP pool provides a competitive 1890
advantage and may help explain the success of Trichodesmium in oligotrophic oceans 1891
characterized by low P concentrations (Sohm and Capone, 2006). 1892
93
The coupled C, N, and P biogeochemical cycles and marine primary production 1893
are central to the regulation of global factors such as atmospheric temperature and surface 1894
ocean pH. Increasing our knowledge of phosphonate metabolism will allow us to 1895
determine potential global implications, such as the production of CH
4
, a potent green 1896
house gas that has been shown to be produced with MnP utilization. This study 1897
addressed the following questions: 1) What are the differences in growth rates in field 1898
populations of Trichodesmium spp. and cultures of Trichodesmium erythraeum IMS101 1899
when grown on either MnP or PO
4
3-
, 2) What are the differences in growth of the 1900
Trichodesmium erythraeum IMS101 associated microbial community (AMC) when 1901
cultures are grown on MnP or PO
4
3-
, 3) Is there preferential use of PO
4
3-
before MnP, and 1902
4) Does MnP utilization result in CH
4
accumulation in field populations of 1903
Trichodesmium spp. and cultures of Trichodesmium erythraeum IMS101? 1904
Materials and Methods
1905
Cultures of Trichodesmium erythraeum IMS101 were maintained in YBCII media 1906
(Chen and Siefert, 2004) with a 12/12 light dark cycle in a Precision low temperature 1907
illuminated incubator (Thermo Scientific, average irradiance 92 µmol photons m
-2
sec
-1
). 1908
Cultures were acclimated to each P source for more than 5 transfers; cultures were then 1909
filtered by gentle filtration, re-suspended in media containing no P source and biomass 1910
was normalize by absorbance before beginning the experiment. Experimental 1911
inoculations included: 1) unfiltered T. erythraeum IMS101 culture, 2) 5 µm filtered T. 1912
erythraeum IMS101 culture which contains only the AMC, and 3) 0.2 µm filtered T. 1913
erythraeum IMS101 culture without T. erythraeum IMS101 or the AMC (abiotic control). 1914
94
The different inoculations and a no inoculation control were maintained in YBCII media 1915
containing the following: 1) no P source control, 2) 10 µM PO
4
3-
, 3) 10 µM MnP, and 4) 1916
5 µM PO
4
3-
and MnP. Three separate replicate bottles for each treatment were used for 1917
daily methane production measurements, while one replicate was reserved for the 1918
remaining experimental measurements. 1919
Field populations of Trichodesmium spp. were collected in the WTNA on board 1920
the R/V Knorr as part of the Amazon influence on the Atlantic: carbon export from 1921
nitrogen fixation by diatom symbioses (ANACONDAS) cruise from May 23 to June 22, 1922
2010 by net tow at a depth of 20 m. The contents of the cod end were placed into a clean 1923
bucket and diluted with 0.2 µm (Supor 200 Membrane Disc Filters, Pall corporation, 1924
USA) filtered surface seawater (FSW). Plastic sterile inoculation loops were used to pick 1925
individual Trichodesmium spp. colonies that were then rinsed three times in FSW. 1926
Samples were then pooled and 10 tufts were placed into 160 mL serum vials containing 1927
150 mL FSW with 20 mM EDTA and either 10 µM MnP or PO
4
3-
additions. Serum vials 1928
were incubated on deck in flow-through incubators shaded to approximate the irradiance 1929
at 20 m. Inoculated FSW with no additional P sources were used as the control. 1930
Biomass was monitored daily using a Turner Designs Trilogy fluorometer, and 1931
optical density at a wavelength of 600 nm (OD
λ600nm
) was used as a proxy for growth. 1932
Methane (CH
4
) accumulation was determined daily by subsampling 100 µL of the 1933
headspace with a gas tight syringe (Hamilton). The concentration was quantified using 1934
gas chromatography (Shimadzu, model GC-mini2) with a flame ionization detector (FID) 1935
after separation on an Alltech HayeSep Q, (80/100 mesh size) column using nitrogen as 1936
95
the carrier gas. Peak heights were determined using a Hewlett Packard integrator (HP 1937
3393A). Standard curves were generated daily by injecting CH
4
gas standards (Matheson 1938
Tri-Gas, 100 ppm CH
4
in nitrogen) of known concentrations at volumes of 100, 50 and 1939
20 µL in triplicate. 1940
Statistical analysis was performed using SigmaPlot’s (Systat Software Inc.) T test 1941
except when assumptions of normality and equal variance were violated resulting in the 1942
use of the non-parametric Mann-Whitney rank sum test to test for identical distributions. 1943
Results 1944
Growth occurred in the unfiltered culture of T. erythraeum IMS101 after one day 1945
in the media containing PO
4
3-
, however growth in the media containing MnP occurred 1946
two days post inoculation (Figure. 1). Growth was greatest in the media containing only 1947
PO
4
3-
on day three (maximum OD
λ600nm
= 0.19), followed by media containing both PO
4
3-
1948
and MnP on day 5 (maximum OD
λ600nm
= 0.15), and media containing only MnP on day 1949
7 (maximum OD
λ600nm
= 0.26). Low growth was evident in the P deplete media, and a 1950
slight increase in optical density was measured on the last day of the experiment 1951
(OD
λ600nm
= 0.01, Figure 1). Growth in treatments containing PO
4
3-
was significantly 1952
greater than in the P deplete control (p < 0.003), however there was no significant 1953
difference in growth among P treatments. 1954
96
1955
Figure
A-‐1.
Absorbance
(600
nm
wavelength)
of
Trichodesmium
erythraeum
IMS101
cultures
1956
grown
in
media
containing
methylphosphonate,
phosphate,
both
methylphosphonate
and
1957
phosphate,
and
a
phosphorus
deplete
control. 1958
Growth curves of the 5 µm filtered cultures of AMC were similar to those 1959
containing T. erythraeum IMS101 and the AMC. Increases in growth occurred after one 1960
day in the media containing PO
4
3-
, and growth in the media containing MnP was evident 1961
two days post inoculation. Growth was greatest in the PO
4
3-
and MnP treatments on day 1962
5 (maximum OD
λ600nm
= 0.22 and 0.18, respectively), and both P sources on day 6 1963
(maximum OD
λ600nm
= 0.18, Figure 2). No growth was evident in P deplete controls, 1964
however slight variations in OD
λ600nm
occurred throughout the experiment (OD
λ600nm
≤ 1965
0.01, Figure 2). Growth in all treatments with a source of P was significantly higher than 1966
P deplete media (p ≤ 0.02). Low growth in the abiotic control occurred in the media 1967
containing PO
4
3-
beginning on day two and continuing until day 6. There was a 1968
97
significant difference in growth of the PO
4
3-
abiotic control on days 7 and 8 (p = 0.002), 1969
which reached its highest value on day 8 (OD
λ600nm
= 0.13, Figure 3). 1970
1971
Figure
A-‐2.
Absorbance
(600
nm
wavelength)
of
the
bacterial
community
associated
with
1972
Trichodesmium
erythraeum
IMS101
cultures
grown
in
media
containing
methylphosphonate,
1973
phosphate,
both
methylphosphonate
and
phosphate,
and
a
phosphorus
deplete
control.
1974
98
1975
Figure
A-‐3.
Absorbance
(600
nm
wavelength)
of
cultures
inoculated
with
0.2
micron
1976
filtered
Trichodesmium
erythraeum
IMS101
cultures
grown
in
media
containing
1977
methylphosphonate,
phosphate,
both
methylphosphonate
and
phosphate,
and
a
phosphorus
1978
deplete
control. 1979
CH
4
concentration in the culture of T. erythraeum IMS101 increased on day 3 in 1980
the media containing MnP, reaching a maximum on day 5 (1.6 ± 0.05 µM CH
4
) followed 1981
by a decrease on day 6 (Figure 4). CH
4
accumulation in the media containing both PO
4
3-
1982
and MnP was an order of magnitude lower and reached a maximum on day 5 (0.16 ± 0.06 1983
µM CH
4
). CH
4
concentrations in P deplete media and the PO
4
3-
treatment remained low 1984
throughout the experiment (≤ 0.08 ± 0.05 µM CH
4
, Figure. 4). CH
4
accumulation was 1985
significantly higher in the MnP treatment (p < 0.001). CH
4
accumulation increased in the 1986
AMC MnP treatment by day 3, and reached a maximum on day 5 (1.6 ± 0.01 µM CH
4
, 1987
Figure 5). CH
4
accumulation with the media containing both PO
4
3-
and MnP reached a 1988
maximum on day 3 (0.22 ± 0.03 µM CH
4
). CH
4
accumulations in P deplete media and 1989
99
the PO
4
3-
treatment remained low throughout the experiment (≤ 0.11 ± 0.05 µM CH
4
, 1990
Figure. 4). CH
4
accumulation was significantly higher in the MnP only treatment (p < 1991
0.001). CH
4
accumulation in the 0.2 µM filtered treatment remained low throughout the 1992
experiment (maximum CH
4
concentrations ≤ 0.28 µM, Figure 6), and no significant 1993
difference was observed among P treatments. 1994
1995
Figure
A-‐4.
Methane
(μM)
production
in
Trichodesmium
erythraeum
IMS101
cultures
1996
grown
in
media
containing
methylphosphonate,
phosphate,
both
methylphosphonate
and
1997
phosphate,
and
a
phosphorus
deplete
control.
1998
100
1999
Figure
A-‐5.
Methane
(μM)
production
in
the
associated
microbial
community
of
2000
Trichodesmium
erythraeum
IMS101
cultures
grown
in
media
containing
methylphosphonate,
2001
phosphate,
both
methylphosphonate
and
phosphate,
and
a
phosphorus
deplete
control.
2002
2003
Figure
A-‐6.
Methane
(μM)
production
in
cultures
inoculated
with
0.2
micron
filtered
2004
Trichodesmium
erythraeum
IMS101
cultures
grown
in
media
containing
methylphosphonate,
2005
phosphate,
both
methylphosphonate
and
phosphate,
and
a
phosphorus
deplete
control.
2006
101
Field populations of Trichodesmium spp. showed similar growth patterns with an 2007
increase in OD
λ600nm
from 0.07 ± 0.002 to 0.08 ± 0.003 on day two followed by 2008
decreasing values through the end of the experiment (Figure 7). Absorbance at the end of 2009
the experiment on day 5 was similar in all P treatments and the control (average OD
λ600nm
2010
= 0.06 ± 0.003, Figure 7). No significant difference in growth among treatments was 2011
observed. CH
4
accumulation in the control varied slightly but remained low with an 2012
initial concentration of 0.99 ± 0.2 µM increasing to 2.1 ± 0.4 µM by the end of the 2013
experiment (Figure 8). Similar CH
4
accumulation occurred in the PO
4
3-
treatment 2014
beginning with 1.10 ± 0.1 µM and increasing to 2.15 ± 0.3 µM by day 5. Initially, CH
4
2015
accumulation in the MnP treatment was similar to the PO
4
3-
treatment and the control 2016
(1.24 ± 0.3 µM), however it rapidly increased and surpassed CH
4
accumulation in the 2017
other treatments by day 2 (6.3 ± 0.3 µM). CH
4
concentrations continued to increase 2018
throughout the experiment reaching a maximum by Day 5 (10.8 ± 0.9 µM, Figure 8). 2019
CH
4
accumulation was significantly higher in the MnP treatment compared to the control 2020
and the PO
4
3-
treatment (p < 0.001). 2021
102
2022
Figure
A-‐7.
Absorbance
(600
nm
wavelength)
of
Trichodesmium
spp.
collected
from
the
WTNA
2023
in
filtered
seawater
containing
methylphosphonate,
phosphate,
and
a
phosphorus
deplete
2024
control.
2025
2026
103
2027
2028
Figure
A-‐8.
Methane
(μM)
accumulation
of
Trichodesmium
spp.
collected
from
the
WTNA
in
2029
filtered
seawater
containing
methylphosphonate,
phosphate,
and
a
phosphorus
deplete
2030
control.
2031
Discussion 2032
Ecological Relevance of MnP and the Marine Methane Cycle 2033
The results of this study suggest the possibility of marine CH
4
production in the 2034
tropical and subtropical oceans. However, further studies will be required to determine if 2035
production was due to Trichodesmium spp. or the AMC. Trichodesmium colonies 2036
collected from the tropical North Atlantic Ocean incubated with an organic phosphorus 2037
compound, methylphosphonate (MnP), resulted in significant CH
4
accumulation. Growth 2038
increased during the first day of incubation, and subsequently decreased in P amendment 2039
treatments and the control (Figure 7). This suggests that the decrease in OD
λ600nm
was not 2040
due to the introduction of the P containing compounds but can be attributed to bottle 2041
104
effects. Significant CH
4
accumulation with the addition of MnP was observed 2042
throughout the experiment (Figure 8). This suggests that Trichodesmium and the AMC 2043
can utilize organic phosphonate compounds as a source of P that can result in the release 2044
of CH
4
. This is consistent with previous studies from station ALOHA in the Pacific 2045
Ocean showing rapid accumulation of CH
4
in unfiltered surface water with the addition 2046
of MnP (Karl et al., 2006). Collectively, these studies demonstrate the utilization of 2047
inorganic MnP by microorganisms across ocean basins that likely play an important role 2048
in the marine CH
4
cycle and contribute to atmospheric CH
4
concentrations. 2049
Metabolic
Efficiency
of
Different
Phosphorus
sources
2050
Culture
studies
were
designed
to
investigate
the
effects
of
MnP
utilization,
2051
specifically
the
metabolic
efficiency
of
growth
on
PO
4
3-
compared
to
MnP,
by
the
2052
marine
diazotroph
T. erythraeum IMS101 and its AMC. Similar growth patterns were 2053
observed between P treatments and different inoculations (Figures 1 and 2). Suggesting 2054
there was not a significant increase in metabolic demand required for MnP utilization. 2055
These results are consistent with previous studies showing T. erythraeum IMS101 was 2056
capable of growth on multiple organic phosphonate compounds with similar metabolic 2057
efficiency (Beversdorf et al., 2010). Another study also found equal growth efficiency on 2058
multiple P substrates, however researchers noted a decrease in P content in cells growing 2059
on organic phosphonates (White et al., 2010). 2060
Preferred Source of Phosphorus 2061
Initial growth rates in the treatments containing PO
4
3-
were similar (Figures 1 and 2062
2). A lag was observed between days 3 and 4 in the treatment containing the AMC and 2063
105
both P sources (Figure 2). This was similar to the lag in initial growth observed in the 2064
MnP only treatment between days 3 and 5. Suggesting that the culture had first utilized 2065
the available PO
4
3-
and then began to utilize the MnP (Figure 2). These results suggest 2066
that PO
4
3-
was utilized before MnP, and was therefore the preferred source of P. Previous 2067
studies showed similar results of simultaneous utilization of PO
4
3-
and MnP along with 2068
the production of greenhouse gasses in cultures of T. erythraeum IMS101 (Beversdorf et 2069
al., 2010). The concurrent release of CH
4
expected with MnP use was not observed in 2070
the treatment containing both P sources. This could be explained by the presence of CH
4
2071
oxidizers in the AMC. These CH
4
oxidizers may have been P limited in the treatment 2072
containing MnP only, which may have resulted in the observed accumulation of CH
4
. 2073
The lack of CH
4
accumulation in the treatment containing both P sources may also be due 2074
to differences in the AMC, as axenic cultures of Trichodesmium IMS101 are unavailable. 2075
Similar CH
4
accumulation patterns were observed in the cultures containing 2076
Trichodesmium IMS101 and the AMC (Figures 4 and 5), suggesting that MnP 2077
degradation and cleavage of the C-P bond was likely due to the AMC. It was not 2078
possible to determine which organisms were ultimately responsible for cleaving the C-P 2079
bond. However, since the CH
4
accumulation patterns were not significantly different 2080
between inoculations, the AMC is thought to be primarily responsible for the 2081
accumulation of CH
4
during this study. Previous studies investigating environmental 2082
Trichodesmium spp. from the Pacific Ocean were found to be associated with bacteria 2083
such as Synechococcus and prochlorococcus, other cyanobacteria (possibly Phormidium), 2084
as well as eukaryotes such as dinoflagellates, diatoms, ciliates, and radiolarians (Hewson 2085
106
et al., 2009). The associated vial community has also been investigated showing an 2086
assorted population of both lytic and temperate phages associated with Trichodesmium 2087
spp. blooms in the Gulf of Mexico (Brown et al., 2013). Further studies need to be 2088
conducted to determine the specific associated microbial community found in cultures 2089
used during this study, and we suggest complementary molecular studies that measure the 2090
expression patterns of phosphonate utilization genes in Trichodesmium IMS101 and the 2091
AMC. 2092
CH
4
cycling in the cultures was also evident as methane increased during the first 2093
part of the experiment suggesting that rates of production were greater then consumption. 2094
CH
4
concentrations decreased after day 5 indicating consumption rates exceeded 2095
production (Figures 4 and 5). Utilization of CH
4
by the AMC was possible in the 2096
treatments containing both source of P. However, the AMC may have been P limited in 2097
the MnP only treatment preventing CH
4
utilization. Mass balance calculations for 2098
methane indicate that the MnP addition was utilized and was approximately equal to the 2099
maximum measured absolute concentration of CH
4
. Utilization of CH
4
may be due to the 2100
presence of methanotrophs in the AMC. However, further studies are needed to identify 2101
the AMC in the specific culture being tested. 2102
We are only beginning to understand the ecological relevance and importance of 2103
the utilization of MnP and other organic compounds in marine systems by specific 2104
species. A recent study has found a member of the ubiquitous Group 1 marine archaea 2105
that produces an exopolysaccharide with MnP attached to the surface, thus providing an 2106
aerobic source of MnP to the worlds oceans (Metcalf et al., 2013). Further studies on the 2107
107
diversity of species capable of utilizing MnP and other DOP compounds are necessary, 2108
and future studies should investigate the abundance, distribution, and expression of 2109
phosphonate utilization genes by marine taxa. Studies determining the half saturation 2110
constants for different marine species and DOP compounds will help to establish the 2111
ecological framework and importance of phosphonates. Identifying sites of the 2112
production of green house gases such as CH
4
in the sea is important and has widespread 2113
implications as CH
4
contributes to greenhouse forcing. Until there is a better 2114
understanding of the availability, concentration, and cycling of the diverse marine 2115
phosphonate compounds we cannot fully understand their importance to biogeochemical 2116
cycles, effects on marine community structure, and the production of CH
4
.
2117
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Abstract (if available)
Abstract
B-vitamins are recognized as important organic growth factors, although our knowledge regarding their concentrations and distribution in aquatic ecosystems is limited. We present the first direct measurements of the organic growth factors thiamin (B₁) and pyridoxine (B₆) in the North Atlantic Ocean that is influenced by Amazon river plume. This is an area known to have high productivity, di-nitrogen (N₂) fixation, and carbon (C) sequestration. The first directly measured vitamin B₆ and biotin (B₇) concentrations from an oligotrophic freshwater system, Lake Tahoe, are also presented. B-vitamins function as essential enzymatic co-factors for diverse biological reactions. Specifically, vitamins B₁ and B₇ are involved in carbon metabolism while vitamin B₆ is required for the metabolism of almost all amino acids. Therefore, vitamins B₁, B₆, and B₇ may play critical roles in both C and nitrogen (N) cycling in aquatic environments as many phytoplankton cannot synthesize these growth factors and need to acquire them from the environment. These studies draw attention to the potential roles of B-vitamins in ecosystem dynamics. ❧ Concentrations of vitamins B₁ and B₆ in the WTNA Ocean ranged from undetectable to 230 and 40 pM, respectively. Depth profiles in the photic zone of B₁ and B₆ varied with depth and salinity. Vitamin B₁ concentrations were significantly higher in the surface plume waters at some stations suggesting a possible riverine influence. Linear regression models were used to determine the influence of vitamins B₁ and B₆ on biologically mediated C and N fixation. The results indicated that the availability of these co-enzymes could affect the rates of these processes in the WTNA. Specifically, significant increases in C and N₂ fixation were observed with increasing concentrations of vitamin B₁ (low salinity and mesohaline stations 9.1 and 1, p value < 0.017 and < 0.03, respectively). A significant positive correlation was also observed between N₂ fixation and vitamin B₁ at station 1 (p value < 0.29) and vitamin B₆ at station 9.1 (p value < 0.017). This study suggests that a dynamic interplay is possible between these organic growth factors and biologically mediated C and N₂ fixation that ultimately affect global biogeochemical cycling. ❧ Concentrations of vitamins B₆ in Lake Tahoe ranged from undetectable to 3.17 and 3.67 pM at the West Lake Tahoe (WLT) and Mid Lake Tahoe (MLT) stations respectively. Vitamin B₇ concentrations ranged from 0.59 to 4.28 pM and 0.23 to 3.45 pM at the WLT and MLT stations, respectively. Other B-vitamins were below the detection limits suggesting that dissolved B-vitamin concentrations in the water column were very low during this study. Generally, the WLT station had higher trace metal concentrations compared to the MLT station suggesting a potential terrestrial source of trace metals to the lake. Depth profiles showed corresponding peaks in trace metals and B-vitamins, and correlation analysis showed a significant relationship of some trace metals and B-vitamins that tended to increase together. This suggests possible trace metal limitation or co-limitation of B-vitamin biosynthesis. ❧ Collectively these studies highlight the importance of B-vitamins to various aquatic systems because of their ability to affect rates of biologically mediated C and N₂ fixation, community structure, and ecosystem functioning. Multiple factors contribute to the abundance and distribution of B-vitamins, specifically species distribution and trace metal concentrations. However, further studies are required to determine the magnitude of the influence of B-vitamins on global biogeochemical cycling and other factors affecting their distribution in various aquatic habitats.
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Creator
Barada, Laila Pualani
(author)
Core Title
The distribution of B-vitamins in two contrasting aquatic systems, and implications for their ecological and biogeochemical roles
School
College of Letters, Arts and Sciences
Degree
Doctor of Philosophy
Degree Program
Biology
Degree Conferral Date
2013-12
Publication Date
08/19/2013
Defense Date
12/19/2013
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
Amazon River Plume,Atlantic Ocean,biogeochemical cycles,biotin,B-vitamins,California,Lake Tahoe,methane,OAI-PMH Harvest,phosphonate,pyridoxine,thiamin,trace metals,Trichodesmium
Language
English
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Electronically uploaded by the author
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Advisor
Capone, Douglas G. (
committee chair
), Fuhrman, Jed A. (
committee member
), Sañudo-Wilhelmy, Sergio A. (
committee member
), Webb, Eric A. (
committee member
), Wilcox, Rand R. (
committee member
)
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lpbarada@gmail.com
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https://doi.org/10.25549/usctheses-c3-321512
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Tags
Amazon River Plume
biogeochemical cycles
biotin
B-vitamins
methane
phosphonate
pyridoxine
thiamin
trace metals
Trichodesmium