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

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

Barada, L. P., Cutter, L., Montoya, J. P., Webb, E. A., Capone, D. G., Sañudo-Wilhelmy, 151

S.A. (2013). The distribution of thiamin and pyridoxine in the western tropical 152

North Atlantic Amazon River plume. Front. Mircrobiol. 4, 1- 10. 153

Benoit, R. J. (1957) Preliminary observation on cobalt and vitamin B
12
in fresh water. 154

Limnol. Oceanogr. 2, 233-240. 155

156

8
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plume-II. Copper, nickel, and cadmium. Deep-Sea Res. 29, 1355–1364. 161

Burkholder, P. R., and Burkholder, L. M. (1958). Studies on B vitamins in relation to 162

productivity of the Bahia Fosforescente, Puerto Rico. Bull. Mar. Sci. 8, 201–223. 163

Capone, D. G., Zehr, J. P., Paerl, H. W., Bergman, B., and Carpenter, E. J. (1997). 164

Trichodesmium, a globally significant marine cyanobacterium. Science 276, 1221– 165

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and development of vitamin-requiring algae. J. Phycol. 5, 64-67. 168

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by phytoplankton. J. Phycol. 6, 351–357. 170

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12
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2
-fixing diatom/cyanobacterial association in 175

the tropical Atlantic Ocean. Mar. Ecol. Prog. Ser. 185, 273–283. 176

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phytoplankton. J. Gen. Microbiol. 16, 286–293. 180

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insights to SAR86, an abundant and uncultivated marine bacterial lineage. ISME J. 183

6, 1186–1199. 184

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in a cosmopolitan oceanic bacterium. Science 309, 1242–1245. 192

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of B-vitamins (B
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12
) and inorganic nutrients on algal bloom dynamics in a 194

coastal ecosystem. Aquat. Microb. Ecol. 49, 181–194. 195

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into Lake Tahoe. Limnol. Oceanogr. 24, 1146-1154 197

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onset of eutrophication in ultra-oligotrophic Lake Tahoe, California-Nevada. 199

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Verhandlungen Internationale Vereinigung Limnologie 27, 7-26 202

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decrease of plastid transketolase activity in antisense tobacco transformants has 204

dramatic effects on photosynthesis and phenylpropanoid metabolism. The Plant 205

Cell 13, 535–551. 206

Jordan, F. (2003). Current mechanistic understanding of thiamin diphosphate-dependent 207

enzymatic reactions. Nat. Prod. Rep. 20, 184–201. 208

Knowles, J. R. (1989). The mechanism of biotin-dependent enzymes. Annu. Rev. 209

Biochem. 58, 195-221 210

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Bacteriol. 37, 259–268. 212

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Tahoe Basin, California and Nevada. Sacramento, CA: U.S. Army Corps of 214

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sea. Limnol. Oceanogr. 11, 621–629. 217

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carboxylases. Arch. Biochem. Biophys. 414, 211-222 219

Ohdake, S. (1932). Isolation of "Oryzanin" (Antineuritic Vitamin) from rice-polishings. 220

(First Report,). Bull. Agr. Chem. Soc. Japan 8, 11-46. 221

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10
Panzeca, C., Tovar-Sanchez, A., Agusti, S., Reche, I., Duarte, C., Taylor, G., and 223

Sañudo-Wilhelmy, S. A. (2006). B vitamins as regulators of phytoplankton 224

dynamics. Eos 87, 593–596. 225

Percudani, R., and Peracchi, A. (2009). The B
6
database: a tool for the description and 226

classification of vitamin B
6
-dependent enzymatic activities and of the 227

corresponding protein families. BMC Bioinformatics 10, 273–281. 228

Pohl, M. (2004). A new perspective on thiamine catalysis. Curr. Opin. Biotechnol. 15, 229

335–342. 230

Provasoli, L., and Pintner, I. J. (1953). Ecological implications of in vitro nutritional 231

requirements of algal flagellates. Ann. N. Y. Acad. Sci. 56, 839–851. 232

Reuter, J. E., S. L. Loeb and C. R. Goldman (1986) Inorganic nitrogen uptake by epilithic 233

periphyton in a N-deficient lake. Limnol. Oceanogr. 31, 149-160. 234

Snell, E. E. (1953). Metabolic significance of B-vitamins. Physiol. Rev. 33, 509–524. 235

Strickland, J. D. H. (2009). Vitamin B
12
, thiamine, biotin. The ecology of the 236

phytoplankton off La Jolla, California, in the period April through September, 237

1967. Bull. Scripps. Inst. Oceanogr. 17, 23–31. 238

Sañudo-Wilhelmy, S. A., Lynda, C., Durazo, R., Smail, E., Gomez-Consarnau, L., Webb, 239

E. A., Prokopenko, M., Karl, D. M., and Berelson, W. M. (2012). Multiple B- 240

vitamin deficiency in large areas of the coastal ocean. Proc. Natl. Acad. Sci. 109, 241

14041-14045. 242

Subramaniam, A., Yager, P., Carpenter, E., Mahaffey, C., Björkman, K., Cooley, S., 243

Kustka, AB, Montoya, J., Sañudo-Wilhelmy, S., and Shipe, R. (2008). Amazon 244

River enhances diazotrophy and carbon sequestration in the tropical North Atlantic 245

Ocean. Proc. Natl. Acad. Sci. 105, 10460–10465. 246

Tovar-Sanchez, A., and Sañudo-Wilhelmy, S. A. (2011). Influence of the Amazon River 247

on dissolved and intra-cellular metal concentrations in Trichodesmium colonies 248

along the western boundary of the sub-tropical North Atlantic Ocean. 249

Biogeosciences 8, 217–225. 250

Voet, D., Voet, J. G., Pratt, C. W. (2001) Fundamentals of Biochemistry. Third edition. 251

New Jersey: Wiley. 449-450 252

Wood, F. E. J. (1966). A phytoplankton study of the Amazon region. Bull. Mar. Sci. 16, 253

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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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

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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

14041-14045. 1749

Schlitzer, R., (2011). Ocean Data View, http://odv.awi.de. 1750

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

Sohm, J.A., Mahaffey, C., Capone, D. G. (2008). Assessment of relative phosphorus 1758

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
nd
edition, 1763

Sunderland, Mass.:Sinauer Associates, Inc. 279-282, 775-776. 1764

Strickland, J. D. H. (2009). Vitamin B12, thiamine, biotin. The ecology of the 1765

phytoplankton off La Jolla, California, in the period April through September, 1967. 1766

Bull. Scripps. Inst. Oceanogr. 17, 23–31. 1767

Subramaniam, A., Yager, P., Carpenter, E., Mahaffey, C., Björkman, K., Cooley, S., 1768

Kustka, AB, Montoya, J., Sañudo-Wilhelmy, S., and Shipe, R. (2008). Amazon 1769

River enhances diazotrophy and carbon sequestration in the tropical North Atlantic 1770

Ocean. Proc. Natl. Acad. Sci. 105, 10460–10465. 1771

Tang, Y. Z., Koch, F., Gobler, C. J. (2010). Most harmful algal bloom species are vitamin 1772

B
1
and B
12
auxotrophs. Proc. Natl. Acad. Sci. 107, 20756–20762. 1773

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A.
A., Agustí,
S.,
Dachs,
J.,
1775

Hutchins,
D.
A.,
Capone,
D.
G.,
Duarte,
C.
M. (2006). Effects of dust deposition and 1776

river discharges on trace metal composition of Trichodesmium spp. in the Tropical 1777

and Subtropical North Atlantic Ocean. Limnol. Oceanogr. 51, 1755-1761. 1778

Tovar-Sanchez, A., and Sañudo-Wilhelmy, S. A. (2011). Influence of the Amazon River 1779

on dissolved and intra-cellular metal concentrations in Trichodesmium colonies 1780

along the western boundary of the sub-tropical North Atlantic Ocean. 1781

Biogeosciences 8, 217–225. 1782

Tyrrell, T. (1999). The relative influences of nitrogen and phosphorus on oceanic primary 1783

production. Nature 400, 525–531. 1784

Van Mooy, B. A. S., Fredricks, H. F., Pedler, B. E., Dyhrman, S. T., Karl, D. M., 1785

Koblížek, M., Lomas, M. W., Mincer, T. J., Moore, L. R., Moutin, T., Rappé, M. S., 1786

Webb, E. A. (2009). Phytoplankton in the ocean use non-phosphorus lipids in 1787

response to phosphorus scarcity. Nature 457, 69–72. 1788

Voet, D., Voet, J. G., Pratt, C. W. (2001). Fundamentals of Biochemistry. Third edition. 1789

New Jersey: Wiley. 449-450. 1790

Wang, D., Aller, R. C., and Sañudo-Wilhelmy, S. A. (2009). A new method for the 1791

quantification of different redox-species of molybdenum (V and VI) in seawater. 1792

Mar. Chem. 113, 250-256. 1793

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assessment of phosphorus and iron physiology in Trichodesmium populations from 1795

the western Central and western South Atlantic. Limnol. Oceanogr. 52, 2221-2232. 1796

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Production of organic matter by Trichodesmium IMS101 as a function of 1798

phosphorus source. Limnol. Oceangr. 55, 1755–1767. 1799

Wood, F. E. J. (1966). A phytoplankton study of the Amazon region. Bull. Mar. Sci. 16, 1800

102–123. 1801

Wu, J., Sunda, W., Boyle, E. A., Karl, D. M. (2000). Phosphate depletion in the western 1802

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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Asset Metadata

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

Contributor Electronically uploaded by the author (provenance)

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:digitallibrary.usc.edu:usctheses,OAI-PMH Harvest,phosphonate,pyridoxine,thiamin,trace metals,Trichodesmium

Language English

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)

Creator Email lpbarada@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-321512

Unique identifier UC11288413

Identifier etd-BaradaLail-2010.pdf (filename),usctheses-c3-321512 (legacy record id)

Legacy Identifier etd-BaradaLail-2010.pdf

Dmrecord 321512

Document Type Dissertation

Rights Barada, Laila Pualani

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

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.