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B-vitamins and trace metals in the Pacific Ocean: ambient distribution and biological impacts

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B-vitamins and trace metals in the Pacific Ocean: ambient distribution and biological impacts

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B-VITAMINS AND TRACE METALS IN THE PACIFIC OCEAN:

AMBIENT DISTRIBUTION AND BIOLOGICAL IMPACTS

by

Emily Ann Smail

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 2012

Copyright 2012 Emily Ann Smail
ii

DEDICATION
I dedicate this dissertation to my family for their constant support and encouragement.

iii

ACKNOWLEDGMENTS
I thank my advisor Sergio Sañudo-Wilhelmy, my co-adviser Eric Webb, and my
committee for their support, encouragement, and dedication to my education. In addition,
I thank Lynda Cutter for her tremendous assistance and Jill Sohm for going out of her
way to assist me. I also thank SeaGrant for my traineeship and funding, the National
Science Foundation for funding, and USC for providing me with fellowship support.
iv

TABLE OF CONTENTS
Dedication ii
Acknowledgments iii
List of Tables vi
List of Figures viii
Abstract xiii
Introduction: The Oceanography of B-vitamins 1
History of Marine B-vitamin Analysis 1
Biological Importance of B-vitamins in the Marine Environment 3
Cycling of B-vitamins in the Marine Environment 6
Research Objectives 7

Chapter One: Longitudinal Gradients of Dissolved Vitamins
(B
6
, B
7
, and B
12
), the Amino Acid Methionine, and the Trace Metal
Cobalt in the Eastern Tropical South Pacific 8
Abstract 8
Introduction 9
Methods 12
Study Site 12
Dissolved B-vitamin and Methionine Sample Collection and
Analysis 13
Dissolved Cobalt Analysis 13
Field Incubation Experiments 14
Nutrient and Flow Cytometry Analysis 14
Chlorophyll and HPLC Pigment Analysis 15
Results 15
Environmental Conditions 15
Vitamin B
12
, Methionine, and Cobalt 21
Vitamin B
6
28
Vitamin B
7
32
Discussion 36
Vitamin B
12
, Methionine, and Cobalt 36
Vitamin B
6
38
Vitamin B
7
40
Summary and Conclusions 41
Chapter One References 43

Chapter Two: The Impact of Thiamine on Nitrogen Fixation by Unicellular
Marine Cyanobacteria 47
Abstract
v

Introduction 48
Methods 49
Thiamin Auxotrophy Conditions and Analysis 49
Field Incubations 51
Culture Nitrogen Fixation Conditions and Analysis 52
Dissolved Thiamin Analysis 53
Results 53
Culture Auxotrophy Analysis 53
Impact of Thiamin on Nitrogen Fixation 54
Dissolved Thiamin Analysis 56
Discussion 57
Chapter Two References 60

Chapter Three: Status of Metal Contamination in Surface Waters of the
Coastal Ocean off Los Angeles, California Since the Implementation of
the Clean Water Act 63
Abstract 63
Introduction 64
Materials and Methods 65
Sample Collection and Study Site 65
Particulate and Dissolved Metal Analyses 67
Phytoplankton Metal Internalization Experiments 67
Results and Discussion 69
Spatial Distribution of Particulate Trace Metals 67
Temporal Gradients in Particulate Trace Metals 71
Potential Sources of Particulate Trace Metals 74
Distribution of Dissolved Trace Metals 75
Temporal Gradients in Dissolved Trace Metals in the
SCB Surface Waters 79
The Case of Lead 81
Rapid Internalization of Trace Metals in Synechococcus sp.
CC9311 82
Chapter Three References 83

Chapter Four: Conclusions and Future Directions 88
Chapter Four References 92

Bibliography 95

Appendices
Appendix A: Supporting Information for Chapter Three 106
vi

LIST OF TABLES

Table i-1: Metabolic roles of select B-vitamins (Madigan et al., 2005). 4

Table 1-1: Concentrations of B
12
and methionine in the Eastern
Tropical South Pacific. Nd = not detected. 25

Table 1-2: Concentrations of vitamin B
6
in the Eastern Tropical South
Pacific. 29

Table 1-3: Concentrations of vitamin B
7
in the Eastern Tropical South
Pacific. 33

Table 2-1: Thiamin biosynthesis genes used for auxotrophy
identification. 50

Table 2-2: Thiamin biosynthesis genes in select unicellular
diazotrophic unicellular cyanobacteria. 53

Table A-1: Approximate coordinates of 1976 near effluent and near
shore stations. 106

Table A-2: Total and % labile particulate trace metal concentrations of
SCB study site surface waters in February 2009. Station
numbers correspond to environmental monitoring stations
by the Los Angeles County sanitation district and the Los
Angeles City sanitation department. 107

Table A-3: Procedural blanks and detection limits for particulate trace
metal analysis. Blanks are the average of filter blanks with
the highest and lowest values not included. Detection limits
are 3x the standard deviation of the blank values. 110

Table A-4: Dissolved trace metal concentrations of SCB study site
surface waters in February 2009. Station numbers
correspond to environmental monitoring stations by the Los
Angeles County sanitation district and the Los Angeles City
sanitation department. 111

Table A-5: Dissolved trace metal concentrations of SCB study site
surface waters in September 2009. Station numbers
correspond to environmental monitoring stations by the Los
Angeles County sanitation district and the Los Angeles City
sanitation department. 113

vii

Table A-6: Procedural blanks and detection limits for dissolved trace
metal analysis. Blanks are the average of Milli-Q® blanks
with the highest and lowest values not included. Detection
limits are 3x the standard deviation of the blank values. 115

Table A-7: Dissolved trace metal concentrations of bi-weekly
samplings in Punta Banda, Mexico (33º N, 117º W) from
2004-2005. 116

viii

LIST OF FIGURES

Figure i-1: Variation in vitamin B
12
concentrations in different marine
environments (Panzeca et al., 2009). 2

Figure i-2: Percentages of eukaryotic algal groups that are known
auxotrophs for vitamins B
12
, B
1
, and B
7
(Croft et al., 2006). 5

Figure 1-1: Map of Eastern Tropical South Pacific sampling stations. 13

Figure 1-2: Nitrate and phosphate concentrations in the Eastern Tropical
South Pacific. 16

Figure 1-3: Dissolved oxygen and temperature in the Eastern Tropical
South Pacific. 16

Figure 1-4: Water masses in the Easter Tropical South Pacific. Water
masses were identified as STW – Subtropical water, SAW –
Subantartic water, ESSW – Equatorial subsurface water,
AIW – Antarctic intermediate water, and PDW – Pacific
deep water. 17

Figure 1-5: Distribution of chlorophyll, heterotrophic bacteria,
Prochlorococcus, Synechococcus, and picoeukaroytes in the
Eastern Tropical South Pacific. 19

Figure 1-6: Phytoplankton composition based on HPLC pigment
analysis at 10m for stations 7 and 11. Pigment signatures for
diatoms-A are based on average values of diatoms grown at
intermediate light, diatoms-B pigments are based on
Pseudo-nitzschia, dinoflagellate-A pigments are based on
average values of dinoflagellates grown at high-light,
dinoflagellate-B pigments are based on average values of
Southern Ocean isolates, haptophyte-A pigments are based
on Emiliania huxleyi isolates, haptophytes B are based on
the average pigment signatures of Phaeocystis isolates, and
cyanobacteria pigment signatures are based on average
values of cyanobacteria grown at highlight (Mackey et al.,
1996). 20

Figure 1-7: Chlorophyll concentrations in control (black) and B
12

(white) treatments at stations 11, 9, and 7. Error bars are
standard deviation of biological replicates. 22

Figure 1-8: Phytoplankton composition based on HPLC pigment
analysis for 48 hour incubations at stations 7 and 11.
ix

Pigment signatures for diatoms-A are based on average
values of diatoms grown at intermediate light, diatoms-B
pigments are based on Pseudo-nitzschia, dinoflagellate-A
pigments are based on average values of dinoflagellates
grown at high-light, dinoflagellate-B pigments are based on
average values of Southern Ocean isolates, haptophyte-A
pigments are based on Emiliania huxleyi isolates,
haptophytes B are based on the average pigment signatures
of Phaeocystis isolates, and cyanobacteria pigment
signatures are based on average values of cyanobacteria
grown at highlight (Mackey et al., 1996). 22

Figure 1-9: Distribution of vitamin B
12
and methionine in the Eastern
Tropical South Pacific. 26

Figure 1-10: Profiles of vitamin B
12
and cobalt in the Eastern Tropical
South Pacific. 27

Figure 1-11: Cobalt and vitamin B
12
concentrations in the Antarctic
(unpublished data), off of Baja California, the North
Atlantic, Long Island Sound (Panzeca et al., 2008, Panzeca
et al., 2009), and the Eastern Tropical South Pacific. Linear
regression and 95% confidence intervals represent
correlations between B
12
and cobalt seen in the upwelling
region off of Baja California. 28

Figure 1-12: Distribution of vitamin B
6
in the Eastern Tropical South
Pacific. 30

Figure 1-13: Linear regression of chlorophyll and B
6
at stations 11, 7, and
9 in the upper transect. Confidence intervals represent 95%
confidence intervals of linear regressions. 31

Figure 1-14: Vitamin and B
6
and chlorophyll at stations profiles and
linear regressions of chlorophyll and B
6
at stations 11, 7,
and 9 in the upper transect. 32

Figure 1-15: Distribution of vitamin B
7
in the Eastern Tropical South
Pacific. 34

Figure 1-16: Chlorophyll concentrations in control (black) and B
12
(white) treatments at stations 11, 9, and 7. Error bars are
standard deviation of biological replicates. 35

Figure 1-17: Phytoplankton composition based on HPLC pigment
analysis for 48 hour incubations at stations 7 and 11.
x

Pigment signatures for diatoms-A are based on average
values of diatoms grown at intermediate light, diatoms-B
pigments are based on Pseudo-nitzschia, dinoflagellate-A
pigments are based on average values of dinoflagellates
grown at high-light, dinoflagellate-B pigments are based on
average values of Southern Ocean isolates, haptophyte-A
pigments are based on Emiliania huxleyi isolates,
haptophytes B are based on the average pigment signatures
of Phaeocystis isolates, and cyanobacteria pigment
signatures are based on average values of cyanobacteria
grown at highlight (Mackey et al., 1996). Nitrate and
phosphate concentrations in the Eastern Tropical South
Pacific. 36

Figure 2-1: Growth of Cyanothece 8902 with thiamin (black) and
without thiamin (red). 54

Figure 2-2: Nitrogen fixation values for control and thiamin addition
incubations calculated as mol N fixed/liter/day in 48 hour
15
N
2
field incubations. Error bars are standard error of
biological replicates. Nitrate and phosphate concentrations
in the Eastern Tropical South Pacific. 55

Figure 2-3: Nitrogen fixation rates in fmol/cell/hour in Cyanothece 8902
and Crocosphaera WH0003 with control (black) and
thiamin (grey) treatments. Error bars are standard deviation
of biological replicates. 56

Figure 2-4: Day (A) and Night (B) profiles of thiamin (black),
photosynthetically active radiation (PAR), and temperature
(blue). Profiles were taken at 24:00 (Day) and 12:00 (Night). 57

Figure 3-1: Map of SCB sampling sites with potential sources of metal
input indicated with arrows. Sampling locations are
identified using the station numbers of the Los Angeles
County Sanitation District and the City of Los Angeles
Sanitation District monitoring programs. Map was generated
using Ocean Data View (Schlitzer, 2011). 66

Figure 3-2: Concentration gradient maps of particulate Cd, Cu, Ba, and
Pb measured in February 2009 in the SCB. The metal
concentration range is indicated with the different colors.
Maps were generated using Ocean Data View (Schlitzer,
2011). 70

xi

Figure 3-3: (A) Particulate metal concentrations measured at the near-
effluent outflow stations in February 1976 (black bars) and
February 2009 (gray bars). The1976 values are mean values
obtained at stations 443 (surface sample) and 361 (10 m off
the bottom) (Bruland and Franks, 1978). The 2009
concentrations are mean ± standard deviation from stations
2802, 2903, and 3504 (Figure 3-1). The location of the 2009
stations was selected based on their proximity to 1976
stations. Station coordinates are available in the Supporting
Information. (B) Box-plots of the enrichment factors (EF)
for particulate metals calculated for all February 2009
stations. The dashed line represents the EF 1 order of
magnitude above what is considered crustal levels (Schiff
and Weisberg, 1999; Wedepohl, 1995). 73

Figure 3-4: Concentration gradient maps of dissolved Ag, Cu, and Cd
measured in February and September 2009 in the SCB. The
metal concentration range is indicated with the different
colors. Maps were generated using Ocean Data View
(Schlitzer, 2011). 77

Figure 3-5: Box-plots of dissolved Cu, Ag, Pb, and Cu concentrations
measured in the SCB in 1989 and in 2009 and in Punta
Banda, Mexico. SCB 1989 concentrations were measured at
near shore stations from Point Loma, Coronado, Imperial
Beach, and the US-Mexico Border (Sañudo-Wilhelmy and
Flegal, 1991, 1992, 1994, 1996). SCB 2009 values are all
the metal concentrations measured in February and
September 2009 in our area of study. Punta Banda
concentrations include all the measurements in samples
collected every 2 weeks from March 2004−April 2005
(Appendix A). The arrows identify specific locations or
oceanographic processes where or when the metal
concentrations were significantly higher. 80

Figure 3-6: Total (gray bars) and intracellular (black bars) Cu and Pb
concentrations measured in Synechococcus sp. CC9311.
Trace metal values are normalized to P to account for
variations in biomass. Error bars are standard error of
culture replicates. 82

Figure A-1: Particulate metal concentrations for near shore stations in
February 1976 (black) and February 2009 (grey). 1976
values are mean values of surface waters from stations 284,
304, 352, 444, 448, 525, and 563 (Bruland & Franks, 1978)
and 2009 stations are mean ± standard deviation of surface
xii

waters samples from stations 2506, 2706, 3306, 4004, and
4001. 2009 stations were selected based on proximity to
1976 stations. 106

Figure A-2: February 2009 surface water particulates for Co, Ni, Fe, V,
Mo, and Zn. 117

Figure A-3: Plot of dissolved and labile particulate Cu from February
2009 samples and dissolved and labile particulate Pb from
February 2009 samples. Error lines represent 95%
confidence interval. 119

Figure A-4: Dissolved trace metal maps of Fe, Ni, V, Mo, and Co for
February and September 2009 stations. 120

Figure A-5: Dissolved Ni, V, Co and Fe in SCB stations and Punta
Banda, Mexico. SCB 1989 Ni and Co are near shore values
from Point Loma, Corando, and Imperial Beach (Sañudo-
Wilhelmy & Flegal, 1996). SCB 2009 values include
February and September samples and Punta Banda values
include samples collected every 2 weeks from March 2004 –
April 2005. 122

Figure A-6: Total (grey) and internal (black) Ni, Co, Fe, Cd, Al, and Zn
in Synechococcus sp. CC9311. Trace metal values are
normalized to phosphorous to account for variations in
biomass. Error bars are standard error of culture replicates. 123

xiii

ABSTRACT
B-vitamins and trace metals have been implicated as important controllers of
phytoplankton abundance and composition in the marine environment. In order to further
establish the distribution and biological importance of dissolved B-vitamin in the Pacific
Ocean, I determined the environmental concentrations of B-vitamins, the vitamin B
12
-
dependent amino acid methionine, and the B
12
-precursor cobalt in the Eastern Tropical
Pacific (ETSP) and the subtropical North Pacific. The environmental relevance of some
toxic trace metals was also established in the coastal ocean off Los Angeles, CA. The
field data was complemented with targeted laboratory and field manipulation experiments
to assist in the interpretation of environmental distributions. In the ETSP, I collected and
analyzed depth-profile measurements of B
12
, methionine, B
7
, B
6
, and cobalt at 6 stations
with environmental conditions ranging from nutrient rich coastal stations to oligotrophic
open ocean stations. Vitamin B
12
and methionine showed similar geographical
distributions suggesting a potential control of vitamin B
12
on the synthesis of the amino
acid likely due to B
12
-dependent methionine synthase. Despite low cobalt levels in the
ETSP (<20pM), vitamin B
12
distribution was only related to the trace metal’s distribution
in a nutrient rich coastal station suggesting that cobalt may only regulate B
12
in locations
where other key macronutrients are plentiful. Vitamin B
6
showed a strong correlation
with chlorophyll indicating that this vitamin may be related to photosynthetic activity.
Vitamin B
7
showed a coastal input and incubation experiments showed that some
phytoplankton may be limited by vitamin B
7
in this region. Large areas of the ETSP were
depleted of B-vitamins and vitamin concentrations were not clearly correlated with
microbial abundance.
xiv

In the subtropical North Pacific, the availability of dissolved thiamin (vitamin B
1
)
was related to nitrogen fixation rates due to the genomically identified thiamin
auxotrophy of abundant group A cyanobacteria. Field B
1
amendment incubation
experiments showed a 46% increase in nitrogen fixation and laboratory culture studies
with an identified B
1
auxotroph showed a 127% increase in nitrogen fixation.
Finally in the Los Angeles coastal ocean, the distribution of dissolved and
particulate trace metals was examined in order to establish current levels of trace metal
contamination in this region of the Pacific Ocean. Particulate levels were shown to be
reduced dramatically compared to levels reported in the 1970s with decreases of ~100-
fold for Pb and ~400-fold for Cu and Cd. Dissolved levels were found to be low with
concentrations within the same range as an uncontaminated site in Punta Banda, Mexico.
A trace metal uptake experiment with Synechococcus sp. CC9311 showed rapid
internalization of multiple metals (within 3 hours) highlighting the importance of
monitoring environmental concentrations of toxic and nutrient metals in the marine
environment.

1

INTRODUCTION: THE OCEANOGRAPHY OF B-VITAMINS

History of Marine B-vitamin Analysis
B-Vitamins were recognized as a required addition to growth media for the
cultivation of many freshwater, brackish, and marine algae beginning in the late 1930’s
(Lwoff & Dusi, 1937). This discovery led to the identification of auxotrophy for the B-
vitamins thiamin (B
1
, C
12
H
17
N
4
OS), biotin (B
7
, C
10
H
16
N
2
O
3
S), and cobalamin (B
12
,
C
63
H
88
CoN
14
O
14
P) in many phytoplankton species and the development of techniques for
measuring the these vitamins in seawater (Carlucci and Silbernagel 1966; Carlucci and
Silbernagel, 1966, 1969; Droop, 1974; Haines and Guillard, 1974). The techniques
originally developed for measuring dissolved concentrations of B-vitamins in seawater
were bioassays that measured the growth response of organisms auxotrophic (requiring
but unable to synthesize) for the vitamin being quantified, thus allowing for indirect
determination of ambient vitamin concentrations (Carlucci and Silbernagel 1966;
Carlucci and Silbernagel, 1966). Recognizing the potential importance of vitamins in
marine ecology, researchers began studying the distributions of B
1
, B
7
, and B
12
in
freshwater and marine environments. Despite this early interest in B-vitamins, the
difficulty of the bioassays prevented studies from being comprehensive enough to fully
estimate the impact of B-vitamins on biological processes (Provasoli and Carlucci, 1974).
As a result, the field suffered a lapse in advancement. However, a recent surge in interest,
led by the development of new direct HPLC quantification methods of B
12
and B
1
as well
as the sequencing of eukaryotic algal genomes, has caused resurgence in the field (Croft
et al., 2005; Croft et al., 2006; Okbamichael and Sanudo-Wilhelmy, 2004, 2005).

2

Figure i-1: Variation in vitamin B
12
concentrations in different marine environments (Panzeca et al., 2009).
The resulting work has focused mainly on: 1) measuring B
12
and B
1

concentrations in the environment; 2) the role of B
12
and B
1
as limiters of phytoplankton
growth; and 3) the metabolic requirements of eukaryotic phytoplankton for B-vitamins
(Bertrand et al., 2007; Croft et al., 2006; Gobler et al., 2007; Panzeca et al., 2008;
Panzeca et al., 2009; Panzeca et al., 2006). It should be noted that these studies have been
performed in few locations at only one or two depths (Bertrand et al., 2007; Gobler et al.,
2007; Panzeca et al., 2009). Based on these studies, vitamin B
12
(Figure 1) and B
1
levels
are generally higher in coastal areas and show seasonal variability with summer months
having lower concentrations (Gobler et al., 2007; Panzeca et al., 2009). This is in contrast
to older bioassay studies that reported that concentrations of B
7
and B
12
were higher in
summer months and significantly lower in winter months (Bruno and Staker, 1978;
Ohwada and Taga, 1972). Studies assessing limitation have shown that picomolar
additions of vitamin B
12
and vitamin B
1
increase phytoplankton productivity and thus are
likely to be among the limiting nutrients in some marine regimes (Bertrand et al., 2007;
3

Panzeca et al., 2006; Sañudo-Wilhelmy et al., 2006). Fitting with these data, a collation
of culture studies found that of 306 phytoplankton species tested, greater than half require
an exogenous supply of vitamin B
12
and approximately a quarter require B
1
(Croft et al.,
2006). These organisms also vary in regard to the metabolic pathways requiring B-
vitamins such as the presence or absence of cobalamin-dependent methionine synthase
and cobalamin-independent methionine synthase (Croft et al., 2006).
The realization of the widespread need for exogenously supplied B-vitamins
among eukaryotic algae, which provide up to 50% of atmospheric carbon fixation (Field
et al., 1998), highlights the importance of studying the oceanography of B-vitamins.
Specifically, there are major knowledge gaps in the distribution and concentrations of B-
vitamins in the oceans and the analysis of biological processes related to B-vitamin
excretion and uptake other primary production.

Biological Importance of B-vitamins in the Marine Environment
B-vitamins are important cofactors for various metabolic pathways including
carbon fixation, fatty acid synthesis, gluconeogenesis, DNA synthesis and repair (Table i-
1; Madigan et al., 2005). While some organisms have the capability to function without
vitamin B
12
there is an absolute requirement for many of the other B-vitamins. For
example, the active form of thiamin (thiamin pyrophosphate) is needed by all phototrophs
for carbon fixation (Schowen, 1998).

4

Table i-1: Metabolic roles of select B-vitamins (Madigan et al., 2005).
B
1
Transketolase, branched chain amino acid synthesis
B
2
Electron transport chains, Redox reactions
B
7
Reverse TCA cycle, fatty acid metabolism, decarboxylations
B
12
Synthesis of deoxyribose, methionine, and carbohydrate
metabolism

The study of B-vitamins in the marine environment began with the discovery that
some eukaryotic algae were B
1
auxotrophs (Lwoff and Dusi, 1937). In addition to B
1
, B
7

and B
12
auxotrophy has also been documented in algae. The requirements for B
1
, B
7
, and
B
12
for algae vary within and across phylogenetic groups as summarized in figure i-2. It
should also be noted that the identification of auxotrophs is likely to be biased by the type
of media used for isolating algal species in culture (Provasoli and Carlucci, 1974). The
presence of auxotrophy in different lineages suggests that it arose independently on
multiple occasions. The simplest explanation for the prevalence of auxotrophy in algal
lineages is that it is the result of a loss of a single biosynthesis gene (Croft et al., 2006).
As it now appears that vitamins are present in many environments (but with unknown
concentrations and undefined fluxes), it is not surprising that auxotrophy is widespread;
thus organisms that do not expend energy synthesizing vitamins could potentially have an
advantage over competitors (Croft et al., 2006; Provasoli and Carlucci, 1974).
As mentioned above B
1
(thiamine) was the first vitamin auxotrophy identified in
algae (Lwoff and Dusi, 1937). The molecule is composed of two moieties, a pyrimidine
and a thiazole moiety, and some algal species have been found to only be auxotrophic for
one of the moieties (Provasoli and Carlucci, 1974). Because thiamin is required by all
known organisms, the algal strains that do not require exogenously supplied thiamin must
5

be synthesizing the compound de novo (Croft et al., 2006). An early study by (Carlucci
and Bowes, 1970a) also observed the algal species Skeletonema costatum and
Stephanopyxis turris secreting thiamin and biotin (B
7
) suggesting that these organisms
are synthesizing these vitamins.
Vitamin B
7
appears to be the least common vitamin auxotrophy studied to date in
eukaryotic algae (Figure i-2). Interestingly, all algal groups with biotin auxotrophy have
complex plastids and also have a requirement for either B
1
, B
12
, or both (Croft et al.,
2006). Biotin is required for crucial metabolic pathways such as the TCA and fatty acid
metabolism and will therefore be required by all phytoplankton (Madigan et al., 2005).
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
B12
B1
B7

Figure i-2: Percentages of eukaryotic algal groups that are known auxotrophs for vitamins B
12
, B
1
, and B
7

(Croft et al., 2006).
Among phytoplankton studied in culture to date, vitamin auxotrophy for B
12
is the
most common (Croft et al., 2006). The synthesis of vitamin B
12
has been well
documented in bacteria and Archaea but has not been verified in eukaryotic algae or other
marine eukaryotes (Raux et al., 2000; Zhang et al., 2009). It seems more likely that algae
that are not auxotrophic for vitamin B
12
have enzymes that do not require B
12
as is the
case for higher plants (Smith et al., 2007). Algal species that do not require exogenously
6

supplied B
12
have also been found to take up the vitamin if it is provided (Bonnet et al.,
2010; Croft et al., 2005). The major enzymes that use vitamin B
12
vary between
organisms with bacteria having over 20 cobalamin-dependent enzymes and eukaryotes
have only two, cobalamin-dependent methionine synthase and methylmalonyl-CoA
mutase (Marsh, 1999).
While the study of B vitamin auxtrophy in marine organisms has focused on
marine phytoplankton, it is important to note that auxotrophy has also been identified in
marine prokaryote (Burkhold and Lewis, 1968). For example, the ubiquitous SAR11
group lacks the synthesis pathways for several universally required vitamins including B
1

and B
7
(Giovannoni et al., 2005). In addition to heterotrophs, it also appears that some
diazotrophic cyanobacteria (UNCY-A) can also be vitamin auxotrophs (Tripp et al.,
2010), further highlighting the linkage between dissolved vitamins and global
biogeochemistry and underscores the importance of establishing the distribution of these
metabolites in the environment.

Cycling of B-vitamins in the Marine Environment
B-vitamins are likely to have major impacts on biogeochemical cycles such as the
carbon cycle do to the important role of phytoplankton in the biological pump (Panzeca et
al., 2008; Panzeca et al., 2006). To fully understand the biogeochemical impacts of these
metabolites, the controls on the production of B-vitamins need to be studied. The
synthesis of B-vitamins will likely correlate with availability of synthesis precursors
including N, P, and Co. One of the precursors for vitamin B
12
synthesis (cobalt, the
central metal in B
12
) has already been shown to limit the production of B
12
(Panzeca et
7

al., 2008). It is likely that the seasonal and regional availability of these precursors to the
organisms that synthesize B-vitamins will regulate their production. There have also been
no studies to date looking at the variations between B-vitamin concentrations and the
abundance of organisms, such as heterotrophic bacteria and cyanobacteria, that are likely
producing them. To fully understand the cycling of B-vitamins in the marine
environment, correlation studies must be done with B-vitamin precursors and producers
to increase the understanding on the controls of B-vitamin production. While correlation
studies are difficult, regional comparison of B-vitamin concentrations correlated to
environmental conditions and community composition will create a starting point for
more in depth culture and mesocosm studies.

Research Objectives
The major objectives of the research presented here are to: 1) establish the
geographical distribution of B-vitamins (B
6
, B
7,
and B
12
), the amino acid methionine, and
cobalt in the Eastern Tropical South Pacific and show how these distributions relate to
physical, chemical, and biological parameters, 2) identify the role of thiamin on nitrogen
fixation of unicellular marine diazotrophs, and 3) determine the current concentrations of
trace metals in the coastal ocean of Los Angeles in order to establish the efficacy of the
implementation of the Clean Water Act. All chapters combine field measurements with
laboratory studies in order to validate environmental trends with culture experiments and
highlight the importance of trace metals and B-vitamins on the biological activity of
marine organisms.
8

CHAPTER ONE: LONGITUDINAL GRADIENTS OF DISSOLVED
VITAMINS (B
6
, B
7,
and B
12
), THE AMINO ACID METHIONINE, AND
THE TRACE METAL COBALT IN THE EASTERN TROPICAL SOUTH
PACIFIC

Abstract
B-vitamins are required coenzymes for many metabolic processes and are
suspected to be important and potentially limiting growth factors in the environment. To
determine the availability of these metabolites for auxotrophic organisms, we completed
depth-profile measurements of B
6
, B
7,
and B
12
in the Eastern Tropical South Pacific
(ETSP), as well as the B
12
-precursor, cobalt, and the B
12
-dependent amino acid,
methionine. Dissolved B-vitamin concentrations were measured at 15 depths at 6
locations ranging from coastal upwelling to open ocean waters, including a location
within the oxygen minimum zone. Our data suggest that the distribution of these vitamins
is dynamic with regard to both depth and station location. Vitamin B
12
concentrations
ranged from 6.99 pM to below detection, methionine ranged from 383.7 pM to below
detection, vitamin B
6
ranged from 8.86 pM to below detection, and vitamin B
7
ranged
from 45 pM to below detection. Vitamin B
7
was the only vitamin that showed a signal in
the oxygen minimum zone. The concentrations of dissolved cobalt were low in the
surface waters (<20 pM), indicating that the availability of this trace element may be a
limiter of B
12
synthesis in some areas. Consistent with their biosynthetic dependence, the
geographical distribution of vitamin B
12
and methionine showed a strong similarity with
each other. Vitamin B
6
showed a strong positive trend with chlorophyll suggesting that
9

this vitamin may play an important role in photosynthesis in the marine environment.
Incubation experiments with additions of B
12
and B
7
showed that some phytoplankton
may be limited by vitamin B
7
, a finding that has not been previously reported. Most
vitamins were undetectable in large oligotrophic regions of the study area, suggesting that
the distribution of these organic compounds may be related to macro nutrient availability
and biological activity.

Introduction
B-vitamins are required for numerous biological activities including carbon
fixation, oxidative stress responses, chlorophyll biosynthesis, amino acid metabolism,
and fatty acid metabolism (Bilski et al., 2000; Croft et al., 2006; Havaux et al., 2009;
Stokstad, 1962; Tsang et al., 2003), making their production and environmental
availability essential to the health of the marine environment. B-vitamins have long been
recognized as being important for the growth of marine phytoplankton that are
auxotrophs for these organic cofactors (Lwoff and Dusi, 1937). B-vitamin auxotrophy in
marine organisms has primarily been identified for vitamins B
1
, B
7
, and B
12
and the
importance of these organic compounds to the growth of numerous phytoplankton has
been well documented (Carlucci and Silbernagel, 1969; Croft et al., 2006; Droop, 1957;
Provasoli and Carlucci, 1974).
To date, B-vitamins have been found to be important not only to environmentally
keystone phytoplankton groups important for carbon export (e.g. diatoms; Croft et al.,
2006; Haines and Guillard 1974) and harmful algal bloom species (e.g. dinoflagelattes;
Tang et al. 2010), but to marine bacteria including the ubiquitous Pelagibacter ubique
10

which appears to be auxotrophic for several vitamins (Croft et al., 2006; Giovannoni et
al., 2005; Haines and Guillard, 1974; Peperzak et al., 2000). These widespread
auxotrophies indicate that the environmental availability of these important metabolites
may control the distribution and activity of many marine organisms.
In order to determine the environmental availability and role of these metabolites
in the environment, recent studies have identified trends including a higher concentration
of some vitamins in some coastal areas, potential colimitation of primary production and
nitrogen fixation by B-vitamins, and an overall dynamic distribution of B-vitamins in the
marine environment including the realization that large areas of the open ocean are
depleted of those organic growth factors (Bertrand et al., 2007; Gobler et al., 2007; Koch
et al., 2011; Panzeca et al., 2009; Panzeca et al., 2006; Sañudo-Wilhelmy et al., 2012;
Sañudo-Wilhelmy et al., 2006). Attention has also been paid to the relationship of the
trace element cobalt, the central metal ion in vitamin B
12
, to the distribution of vitamin
B
12
in the environment with results showing potential limitation of the synthesis of this
vitamin in some select locations (Bertrand et al., 2007; Panzeca et al., 2008; Panzeca et
al., 2009). Work has also been done to identify the potential producers of B
12
in the
marine environment which have been determined to include cyanobacteria and
heterotrophic bacteria (Bertrand et al., 2011; Bonnet et al., 2010; Taylor and Sullivan,
2008). An amino acid, methionine, that is related to vitamin B
12
due to the production of
methionine by B
12
-dependent methionine synthase by many bacteria (Croft et al., 2005),
has also been measured in seawater simultanously with B
12
but only once in a coastal
region off the California-Baja-California coast (Sañudo-Wilhelmy et al., 2012). This
study also included the first ever reported measurements of vitamin B
6
, a cruical organic
11

cofactor that is required for amino acid metabolism, oxidative stress responses, and
chlorophyll synthesis as well as the concentrations of vitamin B
7
which is required for
carboxylase enzymes and fatty acid synthesis (Croft et al., 2006; Havaux et al., 2009;
John, 1995; Stokstad, 1962; Tsang et al., 2003). Methionine, B
12
, B
6
, and B
7
all showed a
dynamic distribution in this Northeast Pacific margin environment (Sañudo-Wilhelmy et
al., 2012), highlighting the importance for further analysis into what controls the
environmental distribution of these metabolites.
Despite the well known importance of these organic growth factors to marine
organisms (Carlucci and Bowes, 1970b; Croft et al., 2006; Droop, 1957; Provasoli and
Carlucci, 1974), depth-profiles and geographical distribution of B-vitamins in different
oceanographic regimes remain sparse (Menzel and Spaeth, 1962; Provasoli and Carlucci,
1974; Sañudo-Wilhelmy et al., 2012). In addition, the relationship of these organic
compounds to other biological and oceangraphic features such as bacterial abundance,
concentrations of macronutrients, and trace metal precursors has not yet been fully
explored. In this study, we report field measurments of several dissolved B-vitamins (B
12
,
B
6
, and B
7
) in two transects from the coastal ocean to the epi-and-mesopelagic zones of
the Eastern Tropical South Pacific (ETSP). Vitamin B
12
analyses were complemented
with analysis of dissolved cobalt and methionine. The study also includes analysis of the
relationship between these B-vitamins to inorganic nutrients (N and P) and other
biological parameters such as microbiological abundance and species composition. To
complement the environmental measurements, we also performed B
12
and B
7
addition
incubation experiments to establish if these vitamins were limiting primary production or
controlling the dominant phytoplankton taxa in the region.
12

Methods

Study Site
Samples were collected at ~15 depths at 6 stations (Figure 1-1) in the Eastern
Tropical South Pacific (ETSP) along a northern (stations 11, 9, and 7) and a more
oligotrophic southern transect (stations 12, 3, and 5). Samples were collected during the
early summer (February 2010 – March 2010) and included sampling in the oxygen
minimum zone of the ETSP. The spatial distribution of primary production in the Eastern
Tropical Pacific has been previously been reported to be generally determined by the
input of nitrate and phosphate from below the thermocline by equatorial upwelling and
wind-driven vertical mixing (Lavin et al., 2006; Pennington et al., 2006). The
phytoplankton community in the coastal zones of the Eastern Pacific had been reported to
be dominated by large celled phytoplankton (diatoms and dinoflagellates) and more
oceanic areas have been found to be dominated by small celled phytoplankton
(Prochlorococcus, Synechococcus, haptophytes, eukaryotic picoplankton, small
dinoflagellates, and pennate diatoms; Pennington et al., 2006).

13

Figure 1-1: Map of Eastern Tropical South Pacific sampling stations.

Dissolved B-vitamin and Methionine Sample Collection and Analysis
Samples were collected using a trace metal clean rosette in acid washed niskin
bottles, filtered in a clean unit through an acid washed 0.22 m capsule filter into
methanol cleaned 1L amber HDPE bottles, and frozen at -20˚C until extraction. Vitamins
and methionine were extracted from seawater samples by solid phase extraction using C
18

resin and analyzed by LC-MS as described in Sañudo-Wilhelmy et al. (2012).

Dissolved Cobalt Analysis
Dissolved samples for cobalt analysis were collected from the same niskin bottles
as dissolved vitamin samples, filtered through 0.22 m capsule filters, and acidified using
Optima grade hydrochloric acid to a pH <2. Samples were stored acidified for at least one
month prior to preconcentration by organic extractions with the APDC/DDDC ligand
14

technique described in Bruland et al. (1985). Cobalt was quantified by ICP-MS using
external calibration curves and an internal indium standard.

Field Incubation Experiments
Incubation experiments were conducted at all stations under trace metal clean
conditions. Water for incubations was prefiltered through 200 m nylon (Nitex) screening
collected from a depth of approximately 10m. Incubations were performed in acid
washed 2L polycarbonate bottles and were filled according to trace metal clean
techniques. Vitamin spike solutions were passed through Chelex as described in Price et
al., 1988/89 to minimize trace metal contamination. Samples were incubated for 48 hours
in a deck board incubator at light and temperature levels set up to mimic in-situ
conditions. Sample treatments included 50 pM B
12
treatments, 500 pM B
7
treatments, and
no addition and control treatments. 500ml of samples were filtered onto GF/F filters and
analyzed for chlorophyll content and analyzed as percentage of control samples. For
stations 7 and 11, the remaining 1500ml of sample was filtered onto GF/F filters and
analyzed by HPLC for pigments (see below).

Nutrient and Flow Cytometry Analysis
Nitrate and nitrite were analyzed according Braman and Hendrix (1989) and
phosphate was analyzed as described in Strickland and Parsons (1968). For flow
cytometry (FC) analysis, samples were fixed with a final concentration of 0.1% formalin
prior to analysis. Samples were run along with an internal standard of BD™ beads on a
FACSCalibur Flow Cytometer. Heterotrophic bacteria were stained with SYTO®13 green
15

fluorescent nucleic acid stain and all FC data was analyzed using the CellQuest software
(BD Biosciences).

Chlorophyll and HPLC Pigment Analysis
Chlorophyll was measured by extracting chlorophyll from GF/F filters in 10ml of
acetone and reading Chla fluorescence on a fluorometer and comparing the readings to
known standards. Environmental chlorophyll was analyzed from 100ml of filtered
seawater and 500ml from incubations. Samples for HPLC pigment analysis were
collected from 1500ml of incubation sample filtered onto GF/F filters. For environmental
pigments, samples were collected at 10m at stations 7 and 11 and 2000ml of sample was
filtered onto GF/F filters. HPLC pigment analysis was performed at Horn Point
Environmental Library and analyzed for species composition with CHEMTAX (Mackey
et al., 1996).

Results

Environmental Conditions
Nitrate + Nitrite concentrations (Figure 1-2) were about an order of magnitude
higher in the upper transect with the lower transect showing oligotrophic conditions at all
stations (average top 50 m – 4.5 M station 11, 4.6 M station 9, and 5.3M station 7
vs. 0.04 M station 12, 0.04 M station 3, and 0.03 M station 5). A similar trend was
also observed for phosphate with concentration in the northern transect being about 2-
times higher than in the more southern locations (Figure 1-2) (average top 100 m – 0.97
16

M station 11, 0.73 M station 9, and 1.0 M station7 vs. 0.34 M station 12, and 0.42 M
station 3, phosphate values unavailable for station 5).
Nitrate + Nitrite ( M)
0 10 20 30 40
Depth (m)
0
200
400
600
800
Phosphate ( M)
0 1 2 3
0
200
400
600
800
Station 11
Station 9
Station 7
Station 12
Station 3
Station 11
Station 9
Station 7
Station 12
Station 3
Station 5

Figure 1-2: Nitrate and phosphate concentrations in the Eastern Tropical South Pacific.
The oxygen minimum zone was present at station 11 from 320m – 550m (DO <5mM/kg)
with low oxygen (DO <5mM/kg) also occurring between 289m – 475m at station 9
(Figure 1-3). The mixed layer depths varied by station with the mixed layer occurring at
~25m at station 11, ~35m at station 9, ~55m at station 7, ~30m at station 12, ~65m at
station 3, and ~75m at station 5 (Figure 1-3).
Dissolved Oxygen (mM/kg)
0 50 100 150 200 250
Depth (m)
0
200
400
600
800
Temperature (ºC)
5 10 15 20 25
0
200
400
600
800
Station 11
Station 9
Station 7
Station 12
Station 3
Station 5
Station 11
Station 9
Station 7
Station 12
Station 3
Station 5

Figure 1-3: Dissolved oxygen and temperature in the Eastern Tropical South Pacific.
17

Water masses influencing the area of study were identified according to temperature and
salinity (Figure 1-4). That analysis shows that the sampling locations and depths were
influenced by the intrusion of several water masses: Subtropical water (STW),
Subantarctic water (SAW), Equatorial subsurface water (ESSW), Antarctic intermediate
water (AIW), and Pacific deep water (PDW). Along the northern transect, stations 7 and
9 were influenced by the same water mass (STW and ESSW), while station 11 has an
intrusion of SAW in the euphotic zone. Along the southern transect, stations 12 and 3
have a strong intrusion of SAW above 300m with the surface waters being predominately
composed of STW. Station 5 has water mass distribution more similar to the northern
transect with the euphotic zone being primarily composed of STW.
STW SAW
AIW
PDW
ESSW
Station 7 Station 9 Station 11
STW
SAW
AIW
PDW
ESSW
Station 5 Station 3 Station 12
% Water mass (%) % Water mass (%)
Longitude (degrees East)
Depth (m) Depth (m)
Longitude (degrees East)

Figure 1-4: Water masses in the Easter Tropical South Pacific. Water masses were identified as STW –
Subtropical water, SAW – Subantartic water, ESSW – Equatorial subsurface water, AIW – Antarctic
intermediate water, and PDW – Pacific deep water.
18

Despite being located between the coastal and the open ocean, bacterial
abundance was highest at station 9 in the northern transect (bacterial maximum of 5.04 x
10
6
cells/ml at station 9) followed by the two coastal stations (bacterial maximum of 1.1 x
10
6
cells/ml at station 11 and 1.4 x 10
6
cells/ml at station 12). The oceanic station on the
northern transect (station 7) had a lower abundance of bacteria followed by stations 3 and
5 along the southern transect (bacterial maximum of 5.79 x 10
5
cells/ml at station 5)
(Figure 1-5). Surface chlorophyll was overall higher along the upper transect with the
highest biomass also found at station 9 (0.18 g/ml at station 11, 0.25 g/ml at station 9,
and 0.17 g/ml at station 7 vs. 0.12 g/ml at station 12, 0.03 g/ml at station 3, and 0.04
g/ml at station 5). The highest chlorophyll depth-profile maximum was also found at
station 9 (1.04 g/ml) and the lowest profile maximum was found at station 3 (0.13
g/ml) (Figure 1-5). The abundance of Synechococcus was highest at station 12 (1.1 x
10
5
cells/ml) and Prochlorococcus was highest at station 5 (7.1 x 10
4
cells/ml).
Picoeukarytoes were most abundant at the two coastal stations with a maximum of 6.9 x
10
3
cells/ml at station 12 and 7.2 x 10
3
cells/ml at station 11.
19

Chl (ug/ml)
0.0 0.2 0.4 0.6 0.8 1.0
Depth (m)
0
50
100
150
Heterotrophic bacteria (cells/ml)
1e+6 2e+6 3e+6 4e+6 5e+6
0
200
400
600
800
Prochlorococcus (cells/ml)
0.0 2.0e+4 4.0e+4 6.0e+4 8.0e+4 1.0e+5 1.2e+5
Depth (m)
0
50
100
150
200
250
Synechococcus (cells/ml)
0 20000 40000 60000
0
50
100
150
200
250
Picoeukaryotes (cells/ml)
0 2000 4000 6000 8000
Depth (m)
0
50
100
150
200
250
Station 11
Station 9
Station 7
Station 12
Station 3
Station 5
Station 11
Station 9
Station 7
Station 12
Station 3
Station 5
Station 11
Station 9
Station 7
Station 12
Station 3
Station 5
Station 11
Station 9
Station 7
Station 12
Station 3
Station 5
Station 11
Station 9
Station 7
Station 12
Station 3
Station 5

Figure 1-5: Distribution of chlorophyll, heterotrophic bacteria, Prochlorococcus, Synechococcus, and
picoeukaroytes in the Eastern Tropical South Pacific.

HPLC pigment analysis at stations 7 and 11 in the northern transects show
variations in the phytoplankton community with station 7 being dominated by
haptophytes with a pigment signature most closely related to Phaeocystis and station 11
being dominated by organisms with pigment signatures related to prasinophytes (Figure
20

1-6). These results are consistent with previous reports (Pennington et al., 2006) with the
coastal station (station 11) having a larger proportion of larger phytoplankton such as
dinoflagellates and the more oceanic station (station 7) having a larger proportion of
smaller phytoplankton such as cyanobacteria and haptophytes.
Prasinophytes
Chlorophytes
Cryptophytes
Diatoms-A
Diatoms-B
Dinoflag-A
Hapto-A
Hapto-B
Dinoflag-B
Cyanobact
% composition
0
10
20
30
40
50
60
Station 7
Station 11

Figure 1-6: Phytoplankton composition based on HPLC pigment analysis at 10m for stations 7 and 11.
Pigment signatures for diatoms-A are based on average values of diatoms grown at intermediate light,
diatoms-B pigments are based on Pseudo-nitzschia, dinoflagellate-A pigments are based on average values
of dinoflagellates grown at high-light, dinoflagellate-B pigments are based on average values of Southern
Ocean isolates, haptophyte-A pigments are based on Emiliania huxleyi isolates, haptophytes B are based on
the average pigment signatures of Phaeocystis isolates, and cyanobacteria pigment signatures are based on
average values of cyanobacteria grown at highlight (Mackey et al., 1996).

In general, the stations showed two-distinct nutrients fields: a more nutrient rich
upper transect and a low nutrient oligotrophic southern transect with two stations (11 and
9) with oxygen minimum zone conditions. Water mass distribution varied by station with
stations 11, 3, and 12 showing an intrusion of SAW where STW dominates the upper
200m at other stations. Biomass (e.g. chlorophyll and bacteria) appears to be overall
21

highest at station 9 in the upper transect and lowest at station 3 in the southern transect.
Prochlorococcus dominated in the most oligotrophic oceanic station (station 5) while
Synechococcus dominated in the two coastal stations (11 and 12).

Vitamin B
12
, Methionine, and Cobalt
The concentrations of vitamin B
12
, methionine, and cobalt measured in this study
are presented in table 1-1. Concentrations of vitamin B
12
and methionine were highest at
stations 11 and 7 in the upper transect and were largely undetectable along the southern
transect and at station 9 in the upper transect though the region does not appear to be B
12

limited (see incubation results below). Along the upper transect, vitamin B
12
ranged from
below detection to 6.99 pM at station 7, below detection to 0.10 pM at station 9, and
below detection to 6.87 pM at station 11. Similar to the geographical distribution of
vitamin B
12
, methionine ranged from below detection to 383.7 pM at station 7, below
detection to 2.13pM at station 9, and below detection to 379.9 pM at station 11. Along
the southern transect, concentrations reached a maxima of 0.17 pM for B
12
and 4.96 pM
for methionine (Figure 1-9). Despite low to undetectable concentrations of vitamin B
12
at
many of the stations, B
12
incubation experiments did not show a significant increase in
chlorophyll content with B
12
additions at station 7, 9, or 11 (Figure 1-7) or a significant
change in species composition based on pigment analysis at stations 7 and 11 (Figure 1-
8). This suggests that phytoplankton at those open ocean and coastal locations were not
B
12
-limited during our study.
22

Station 11 Station 9 Station 7
Chl ( g/ml)
0
50
100
150
200
250

Figure 1-7: Chlorophyll concentrations in control (black) and B
12
(white) treatments at stations 11, 9, and
7. Error bars are standard deviation of biological replicates.
Prasinophytes
Chlorophytes
Cryptophytes
Diatoms-A
Diatoms-B
Dinoflag-A
Hapto-A
Hapto-B
Dinoflag-B
Cyanobact
% composition
0
10
20
30
40
50
60
Station 7 Control
Station 7 B
12
Prasinophytes
Chlorophytes
Cryptophytes
Diatoms-A
Diatoms-B
Dinoflag-A
Hapto-A
Hapto-B
Dinoflag-B
Cyanobact
% composition
0
10
20
30
40
50
60
Station 11 Control
Station 11 B12

Figure 1-8: Phytoplankton composition based on HPLC pigment analysis for 48 hour incubations at
stations 7 and 11. Pigment signatures for diatoms-A are based on average values of diatoms grown at
intermediate light, diatoms-B pigments are based on Pseudo-nitzschia, dinoflagellate-A pigments are based
on average values of dinoflagellates grown at high-light, dinoflagellate-B pigments are based on average
values of Southern Ocean isolates, haptophyte-A pigments are based on Emiliania huxleyi isolates,
haptophytes B are based on the average pigment signatures of Phaeocystis isolates, and cyanobacteria
pigment signatures are based on average values of cyanobacteria grown at highlight (Mackey et al., 1996).

The distribution of vitamin B
12
and methionine was dynamic in the ETSP and
could potentially be related to the different availability of macronutrients and biological
activity in the two transects (Figure 1-2 and 1-5), as those two organic compounds were
23

undetectable in the low nutrient- low biomass lower transect. One exception to this trend
was station 9 in the northern transect where despite relatively high nutrients, vitamin B
12

and methionine were also virtually undetectable. At stations where the organic
compounds were detectable, the vertical distribution of methionine and B
12
showed
maxima at mid-depths in the euphotic zone (6.99 pM at 20m for vitamin B
12
and 383.7
pM at 20m for methionine at station 7; 6.9 pM at 60m for vitamin B
12
and 379.9 pM at 60
for methionine at station 11). A clear relationship between chlorophyll concentrations and
heterotrophic and autotrophic bacterial abundance or water mass distribution with the
concentration of methionine and vitamin B
12
was not observed (Figures 1-4, 1-5, and 1-
9). For example, at station 9 where chlorophyll and bacterial biomass were high, vitamin
B
12
and methionine were undetectable in contrast to the station with the second highest
chlorophyll and bacterial abundance (station 11) where the concentrations of B
12
and
methionine were the high.
25
Table 1-1: Concentrations of B
12
and methionine in the Eastern Tropical South Pacific. Nd = not detected.
(Oceanic) Upper transect (Coastal) (Oceanic) Lower transect (Coastal)
Station 7 Station 9 Station 11 Station 5 Station 3 Station 12
Depth
(m)
B
12

(pM)
Met
(pM)
Depth
(m)
B
12

(pM)
Met
(pM)
Depth
(m)
B
12

(pM)
Met
(pM)
Depth
(m)
B
12

(pM)
Met
(pM)
Depth
(m)
B
12

(pM)
Met
(pM)
Depth
(m)
B
12

(pM)
Met
(pM)
10
1.04 ±
0.02
180.11
± 0.3 10 nd nd 10
1.98 ±
0.42
166.66
± 28 10 nd
4.8 ±
0.87 10
0.17 ±
0.00
3.47
±
2.37 10 nd nd
20
6.99 ±
0.83
383.7
± 1.0 20 nd
2.13 ±
0.05
50

6.31 ±
0.82
378.1 ±
24 40 nd
0.72 ±
0.02 40 nd nd 50 nd
4.96
±
6.41
40
5.57 ±
0.03
263.55
± 5.6 35
0.10
±
0.18
0.23 ±
0.01 60
6.9 ±
0.61
379.94
± 33 70
0.02 ±
0.00
0.04 ±
0.05 60 nd
1.17
±
0.22 80 nd nd
70
2.09 ±
0.01
161.5
± 1.9 80 nd
0.10 ±
0.15 120
6.87 ±
0.48
219.98
± 11 100
0.28 ±
0.5
0.49 ±
0.69 100 nd nd 200 nd nd
80
2.33 ±
0.37
197.5
± 1.9 95
0.03
±
0.05
0.1 ±
0.01 250
3.75 ±
0.64
82.5 ±
1.5 140 nd
0.03 ±
0.00 150 nd nd 250 nd nd
95
0.57 ±
0.04
51.3 ±
2.3 250 nd nd 360
0.08 ±
0.01
49.6 ±
1.6 400 nd nd 180 nd nd 340 nd nd
130
0.2 ±
0.01
69.6 ±
1.1 300 nd nd 400
0.11 ±
0.03
49.75 ±
6.6 500
0.13
± 0.24
0.49 ±
0.07 200 nd nd 400 nd nd
200 nd
61.9 ±
4.5 325 nd nd 700 nd nd 700 nd nd 250 nd nd 500 nd nd
300 nd
28.9 ±
1.4 400 nd nd 1000 nd nd 1000 nd nd 300 nd nd 550 nd nd
400 nd
18.8 ±
2.8 500 nd nd 2000
0.33 ±
0.57
0.51 ±
0.72 350 nd nd 1000 nd nd
700 nd nd 700 nd nd 3000 nd nd 500 nd
1000 nd nd 1000 nd nd 700 nd
1000

26
The geographical distribution of methionine and vitamin B
12
concentrations
showed overall similar trends in the upper transect between the two compounds (Figure
1-9), while both compounds were mostly undetectable in the lower transect. For stations
11 and 7 where B
12
and methionine were abundant (6.99 pM – below detection B
12
and
383.7 pM – below detection methionine at station 7 and 6.87 pM – below detection B
12

and 379.7 pM – below detection methionine at station 11), the correlation coefficients
were r
2
= 0.87 at station 7 and r
2
= 0.72 at station 11.
B
12
(pM)
0 2 4 6 8
Depth (m)
0
200
400
600
800
Methionine (pM)
0 100 200 300 400
Station 11
Station 9
Station 7
Station 3
Station 5
Station 12
Station 11
Station 9
Station 7
Station 3
Station 5
Station 12

Figure 1-9: Distribution of vitamin B
12
and methionine in the Eastern Tropical South Pacific.

Cobalt concentrations in the upper 60m, where the highest levels of vitamin B
12

were detected, ranged from 19 – 40 pM at station 11, 8 – 22 pM at station 9, 6 – 11 pM at
station 7, 12 –7 pM at station 12, 9 –13 pM at station 3, and 9 – 10 pM at station 5
(Figure 1-10). Vitamin B
12
was largely undetectable along the southern transect where
cobalt levels and nutrient levels were of a lower range (7.0 pM to 46 pM Co along the
lower transect vs. 6.3 pM to 84 pM Co along the northern transect). Vertical distributions
of cobalt and vitamin B
12
showed similar trends at station 11 where low B
12

concentrations were found at the surface where low cobalt was found and B
12
maxima
27

values occurring at depths where cobalt concentrations showed increases (Figure 1-10).
This trend between cobalt and B
12
was not observed at other stations.
Co (pM)
0 20 40 60 80
Depth (m)
0
200
400
600
800
B
12
(pM)
0 2 4 6 8
0
200
400
600
800
Cobalt (pM)
0 20 40 60 80 100
Station 11 B
12
Station 11 Cobalt
A
B

Figure 1-10: Profiles of vitamin B
12
and cobalt in the Eastern Tropical South Pacific.
Compared to the relationship between cobalt and vitamin B
12
observed at other
locations (Antarctica, Baja California, and the North Pacific; (Panzeca et al., 2008;
Panzeca et al., 2009), vitamin B
12
concentrations in the ETSP were found at lower cobalt
levels (Figure 1-11). This trend is especially clear among values in top 40m of the upper
transect where vitamin B
12
was measurable despite <20 pM cobalt concentrations
(station 7 at 20m = 6.99 pM B
12
and 8.9 pM Co, station 7 at 40m = 5.57 pM B
12
and 6.3
pM Co; station 11 at 10m = 1.9 pM B
12
and 19.2 pM Co). Among previously reported
B
12
and cobalt concentrations, a noticeable correlation between cobalt and B
12
has only
been observed in the upwelling region of Baja California (r
2
= 0.37; Figure 1-11). As a
relationship between cobalt and B
12
was only observed at the coastal, relatively nutrient
rich station 11 in the ETSP, it is possible that cobalt concentrations only control the
distribution of B
12
in areas where other essential nutrients such as nitrogen and
phosphorous are readily available.
28

Dissolved Co (pM)
0 200 400 600 800 1000 1200 1400 1600 1800 2000
Dissolved B
12
(pM)
0
5
10
15
20
25
Antarctic
Upwelling coastoff Baja California
Upwelling-linear regression
Upwelling-95% Confidence limits
Todos Santos Bay, Baja California
North Atlantic Ocean (Azores to Iceland)
Long Island Sound (Spring)
Long Island Sound (Summer)
ETSP

Figure 1-11: Cobalt and vitamin B
12
concentrations in the Antarctic (unpublished data), off of Baja
California, the North Atlantic, Long Island Sound (Panzeca et al., 2008, Panzeca et al., 2009), and the
Eastern Tropical South Pacific. Linear regression and 95% confidence intervals represent correlations
between B
12
and cobalt seen in the upwelling region off of Baja California.

Vitamin B
6

Along the upper transect, concentrations of vitamin B
6
ranged from below
detection to 4.27 pM at station 7, below detection to 3.95 pM at station 9, and below
29

detection to 5.27 pM at station 11 (Table 1-2 and Figure 1-12). Depth distributions
showed maxima levels at mid-depths in the euphotic zone (20-80m) at all stations in the
upper transect. Along the lower transect, concentrations of vitamin B
6
ranged from below
detection to 4.12 pM at station 5, below detection to 8.86 pM at station 3, and below
detection to 3.19 pM at station 12. Concentrations for all other depths for B
6
are
presented in table 1-2.
Table 1-2: Concentrations of vitamin B
6
in the Eastern Tropical South Pacific.
(Oceanic) Upper transect (Coastal) (Oceanic) Lower transect (Coastal)
Station 7 Station 9 Station 11 Station 5 Station 3 Station 12
Dept
h
(m)
B
6

(pM)
Depth
(m)
B
6

(pM)
Dept
h
(m)
B
6

(pM)
Dept
h (m)
B
6

(pM)
Dept
h
(m)
B
6

(pM)
Dept
h
(m)
B
6

(pM)
10
1.04 ±
0.01 10 nd 10
1.67 ±
0.06 10
4.12 ±
0.68 10
3.57 ±
0.00 10 nd
20
3.25 ±
0.23 20
3.95 ±
2.14 50
5.27 ±
0.18 40
0.02
±0.003 40
4.47 ±
2.70 50 nd
40
2.95 ±
0.03 35
0.28 ±
0.25 60
4.16 ±
0.31 70 nd 60
0.34 ±
0.07 80 nd
70
3.2 ±
0.02 80
1.60 ±
0.93 75
3.02 ±
0.31 100 nd 100 nd 200
3.19 ±
1.30
80
4.27 ±
0.29 95
1.35
±1.45 100 nd 140 nd 150
8.86 ±
1.88 250
0.83
±0.84
95
3.48 ±
0.21 250
0.10 ±
0.01 120 nd 200 nd 180
0.30 ±
0.05 340
2.41 ±
3.01
130
0.85 ±
0.07 300
0.33 ±
0.09 200 nd 400 nd 200
4.66
±3.46 400 nd
200 nd 325 nd 250 nd 500 nd 250
5.47 ±
0.96 500
2.61 ±
2.22
300 nd 400
0.03 ±
0.05 300 nd 700 nd 300
1.73 ±
1.51 550 nd
400 nd 500
2.65 ±
1.29 360 nd 1000 nd 350 nd 1000 nd
700 nd 700
0.96 ±
1.3 380 nd 2000 nd 500 nd
1000 nd 1000
0.28 ±
0.25 400 nd 3000 nd 700 nd
700 nd 1000 nd
1000 nd

Depth profile distributions were more variable along the lower transect with
maxima values found at 10m at station 5, 150m at station 3, and 200m at station 12
(Figure 1-12). The geographical distributions of this B-vitamin show a possible coastal
30

inputs at station 12 in the southern transect. The geographical distribution for this B-
vitamin does not appear to vary with nutrient concentrations with the highest
concentrations found at oligotrophic station 3 (8.86 pM at 150m and 5.47 pM at 250m)
and the relatively nutrient rich station 11 (5.27 pM at 50m) (Figure 1-12, table 1-2), and
does not appear to follow water mass distributions.
B
6
(pM)
0 2 4 6 8 10 12
Depth (m)
0
200
400
600
800
Station 11
Station 9
Station 7
Station 3
Station 5
Station 12

Figure 1-12: Distribution of vitamin B
6
in the Eastern Tropical South Pacific.
One striking trend in the distribution of vitamin B
6
was the correlation with
chlorophyll (Figures 1-13 and 1-14) in the more nutrient rich upper transect. While a
correlation was observed between B
6
and chlorophyll with all data pooled in the upper
transect (Figure 1-13, r
2
= 0.43), the correlation was notably stronger when B
6
and
chlorophyll were separated by station (r
2
= 0.96 station 11, r
2
= 0.58 station 9, r
2
= 0.66
31

station 7). This trend was not observed in the southern transect (stations 3, 5, and 12).
Vitamin B
6
levels in the southern transect also did not show a trend of tracking bacterial
abundance among heterotrophic bacteria or cyanobacteria (Figure 1-5).
B
6
(pM)
0 1 2 3 4 5 6
Chl ( g/ml)
0.0
0.2
0.4
0.6
0.8
r
2
= 0.43

Figure 1-13: Linear regression of chlorophyll and B
6
at stations 11, 7, and 9 in the upper transect.
Confidence intervals represent 95% confidence intervals of linear regressions.
32

Chl ( g/ml)
0.0 0.2 0.4 0.6 0.8 1.0 1.2
0
20
40
60
80
100
120
140
B
6
(pM)
0 1 2 3 4 5 6 7
Station 9
Chl ( g/ml)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Depth (m)
0
20
40
60
80
100
120
140
B
6
(pM)
0 1 2 3 4 5 6
Station 11
Chlorophyll ( g/ml)
0.0 0.1 0.2 0.3 0.4 0.5
Depth (m)
0
20
40
60
80
100
120
140
B
6
(pM)
0 1 2 3 4 5
Station 7

Figure 1-14: Vitamin and B
6
and chlorophyll at stations profiles and linear regressions of chlorophyll and
B
6
at stations 11, 7, and 9 in the upper transect.

Vitamin B
7

Concentrations of vitamin B
7
along the upper transect ranged from below
detection to 18.36 pM at station 7, below detection to 0.71 pM at station 9, and below
detection to 45 pM at station 11 (Table 1-3 and Figure 1-15). Along the lower transect,
vitamin B
7
concentrations were below detection at stations 3 and 5 and ranged from
below detection to 4.20 pM at station 12. The vertical distribution

33

Table 1-3: Concentrations of vitamin B
7
in the Eastern Tropical South Pacific.
(Oceanic) Upper transect (Coastal)
(Oceanic) Lower transect
(Coastal)
Station 7 Station 9 Station 11 Station 5 Station 3 Station 12
Depth
(m)
B
7

(pM)
Depth
(m)
B
7

(pM)
Depth
(m)
B
7

(pM)
Depth
(m)
B
7

(pM)
Depth
(m)
B
7

(pM)
Depth
(m)
B
7

(pM)
10
14.78
± 0.17 10 nd 10
44.46
± 5.0 10 nd 10 nd 10 nd
20
18.36
± 2.27 20 nd 50
39.6 ±
1.8 40 nd 40 nd 50 nd
40
11.53
± 0.32 35 nd 60
40.2 ±
1.7 70 nd 60 nd 80 nd
70
10.5 ±
0.03 80 nd 75
28.5 ±
1.9 100 nd 100 nd 200 nd
80
5.41 ±
0.90 95 nd 100
45.0 ±
1.8 140 nd 150 nd 250 nd
95
3.13 ±
0.30 250 nd 120
27.9 ±
0.6 200 nd 180 nd 340
3.99
±
3.53
130
1.23 ±
0.19 300
0.14 ±
0.13 200
26.0 ±
1.2 400 nd 200 nd 400 nd
200
0.17 ±
0.07 325 nd 250
27.9 ±
0.06 500 nd 250 nd 500
4.20
±
3.64
300 nd 400
0.027 ±
0047 300
7.3 ±
0.3 700 nd 300 nd 550 nd
400 nd 500
0.71 ±
0.62 360
26.7 ±
1.17 1000 nd 350 nd 1000 nd
700 nd 700 nd 380
28.5 ±
1.3 2000 nd 500 nd
1000 nd 1000 nd 400
28.28
± 1.7 3000 nd 700 nd
700
13.9 ±
0.7 1000 nd
1000 nd

of vitamin B
7
varied by station with maximum values occurring in the euphotic zone for
stations 7 and 11 and below the euphotic zone at stations 9 and 12 (Figure 1-15). Vitamin
B
7
was also detected in the oxygen minimum zones of stations 9 and 11. Below, 500m no
vitamin B
7
was detected in the lower transect and in the upper transect were only found
below 500m at the coastal station on the upper transect (700m – station 11). This vitamin
was, in general, found in the euphotic zone within the top 200m ranging from below
detection to 45pM and below detection to below detection to 28.28 pM below that depth.
34

The geographical distribution of vitamin B
7
showed a strong coastal input (Figure 1-15)
in both the upper and lower transect with the highest concentrations along the two
transects being found at stations 11 (45pM) and station 12 (4.20pM). It should also be
noted, that at station 11 there appears to be a spike in vitamin B
12
in the oxygen minimum
zone (B
7
>26 pM at 360-400m, OMZ present from 320-500m).
B
7
(pM)
0 10 20 30 40 50 60
Depth (m)
0
200
400
600
800
Station 11
Station 9
Station 7
Station 3
Station 5
Station 12

Figure 1-15: Distribution of vitamin B
7
in the Eastern Tropical South Pacific.
The distribution of vitamin B
7
did not show a clear geographical correlation with
bacterial abundance, chlorophyll concentrations, or water mass distribution (Figures 1-4
and 1-5). However, a significant average increase in chlorophyll of 29% (p-value = 0.02)
was observed with the addition of vitamin B
7
to incubation experiments at station 7
indicating potential limitation of primary production by this vitamin in this area. A
significant increase in chlorophyll was not observed in incubation experiments at other
stations (Figure 1-16).
35

Station 11 Station 9 Station 7
Chl ( g/ml)
0
50
100
150
200
250

Figure 1-16: Chlorophyll concentrations in control (black) and B
12
(white) treatments at stations 11, 9, and
7. Error bars are standard deviation of biological replicates.

Pigment analysis of incubation experiments showed a stimulation in the growth of
haptopytes with a pigment signature most closely related to that of Phaeocystis at station
7 (average increase of 33%, p-value = 0.05). This trend was not observed at station 11
where B
7
additions did not cause a significant change in the pigment composition of the
phytoplankton community relative to the control (Figure 1-17).
36

Prasinophytes
Chlorophytes
Cryptophytes
Diatoms-A
Diatoms-B
Dinoflag-A
Hapto-A
Hapto-B
Dinoflag-B
Cyanobact
% composition
0
10
20
30
40
50
60
Station 7 Control
Station 7 B
7

Prasinophytes
Chlorophytes
Cryptophytes
Diatoms-A
Diatoms-B
Dinoflag-A
Hapto-A
Hapto-B
Dinoflag-B
Cyanobact
% composition
0
10
20
30
40
50
60
Station 11 Control
Station 11 B
7

Figure 1-17: Phytoplankton composition based on HPLC pigment analysis for 48 hour incubations at
stations 7 and 11. Pigment signatures for diatoms-A are based on average values of diatoms grown at
intermediate light, diatoms-B pigments are based on Pseudo-nitzschia, dinoflagellate-A pigments are based
on average values of dinoflagellates grown at high-light, dinoflagellate-B pigments are based on average
values of Southern Ocean isolates, haptophyte-A pigments are based on Emiliania huxleyi isolates,
haptophytes B are based on the average pigment signatures of Phaeocystis isolates, and cyanobacteria
pigment signatures are based on average values of cyanobacteria grown at highlight (Mackey et al., 1996).

Discussion

Vitamin B
12
, Methionine, and Cobalt
Production of vitamin B
12
is only known to occur in prokaryotes (Croft et al.,
2005; Zhang et al., 2009) and as such we were expecting the stations with higher
heterotrophic and autotrophic bacteria to have higher concentrations of this vitamin.
While this trend was observed at stations 7 and 11, a clear exception to this trend was
station 9 located in the northern transect which had the highest overall bacterial
abundance and primarily undetectable vitamin B
12
. One possibility explanation for the
low B
12
levels at this station is that the phytoplankton were stripping vitamin B
12
from the
dissolved pool leading to low ambient concentrations, as the chlorophyll levels at this
37

station were also the highest measured at all locations. Despite the overall low levels of
B
12
at this station, vitamin B
12
addition experiments did not show a significant increase in
chlorophyll, and vitamin additions did not generate a significant shift in the
phytoplankton pigments detected at stations 7 and 11. This suggests that phytoplankton in
this region were not limited by the availability of vitamin B
12
.
One important factor for explaining the distribution of vitamin B
12
in the ETSP is
the availability of cobalt, the central metal ion in the vitamin. Depth distribution trends
were overall similar to those previously reported (Saito and Moffett, 2002; Saito et al.,
2004; Saito et al., 2005). However, the concentrations were overall lower than those
previously reported for the Peru upwelling region (27 – 315 pM cobalt in the Peru
upwelling vs. 6.35 – 101 pM in the ETSP) (Saito et al., 2004). Previous studies have
shown that cobalt can be a limiting trace element for B
12
synthesis at low (<20 pM)
concentrations (Panzeca et al., 2008). Cobalt levels of less than 20 pM were detected in
the upper layers of the water column of all stations in the ETSP. The lower transect
cobalt levels were lowest with all stations in the lower transect having cobalt
concentrations less than 20 pM down to a depth of 100-250m which could be related to
the undetectable B
12
at these stations. The only station where cobalt levels were
consistently above 20pM in the top 60m was station 11. This station was also the only
station that showed a depth-profile relationship between B
12
and cobalt. Although we did
not do experiments to establish how cobalt amendments could increase the synthesis of
B
12
, our results suggest that cobalt levels could be limiting the production of vitamin B
12

in this region. It is also possible that low nitrogen and phosphorous levels at many of the
38

stations could be leading to a co-limitation with cobalt for the production of vitamin B
12

in areas of the ETSP.
Aside from the direct requirement of vitamin B
12
by some marine organisms, our
data suggest vitamin B
12
may also be an important controller of methionine distribution.
Methionine showed a similar distribution to vitamin B
12
which we hypothesize is the
result of a link between vitamin B
12
and methionine due to the activity of B
12
-dependent
methionine synthase. Understanding what controls the distribution of methionine is of
interest as many important marine organisms such as UCYN-A cyanobacteria and the
ubiquitous Pelagibacter ubique have been shown to require an exogenous supply of
methionine (Tripp et al., 2010; Tripp et al., 2008). We suggest that future studies explore
this relationship further in order to better understand the cycling of this amino acid in the
marine environment.

Vitamin B
6

In contrast to concentrations reported off of the California-Baja California border,
vitamin B
6
was detected at all stations. In the upper transect of the ETSP where
chlorophyll levels were highest, a strong correlation between vitamin B
6
and chlorophyll
was observed (Figures 1-5 and 1-12). This relationship suggests that this vitamin may be
primarily synthesized by autotrophic phytoplankton. B
6
is a crucial cofactor required for
both the synthesis of amino acids, oxidative stress responses, and chlorophyll synthesis
(Havaux et al., 2009; Stokstad, 1962; Tsang et al., 2003). It is possible that phytoplankton
require high amounts of this vitamin for photosynthetic activity. As vitamin B
6
has been
shown to be protective against high light and oxidative stress (Havaux et al., 2009), it is
39

also possible that B
6
synthesis could be important to marine phytoplankton in response to
that type of environmental stress. Variations in the B
6
-chlorophyll correlations observed
at the different locations in the upper transect may be related to differences in
phytoplankton composition. For example, station 7 was dominated by haptophytes while
station 11, that had a higher correlation between B
6
and chlorophyll, was dominated by
prasinophytes (Figure 1-6). Further research is needed to determine if production of
vitamin B
6
varies by phytoplankton groups including eukaryotic phytoplankton groups
and cyanobacteria.
In contrast to the northern transect, the distribution of vitamin B
6
in the southern
transect appears to be controlled by something other than phytoplankton abundance. This
could potentially be explained by differences in the phytoplankton community between
the upper and lower transect with the low nutrient southern transect being expected to be
dominated by small phytoplankton which has been reported at oligotrophic stations in the
Eastern Tropical Pacific (Pennington et al., 2006). In contrast to the distribution of
vitamin B
12
, vitamin B
6
is above detection in the most of the oligotrophic southern
transect. One possible explanation is that vitamin B
6
is less metabolically expensive than
vitamin B
12
(C
63
H
88
CoN
14
O
14
P) as B
6
(C
8
H
11
NO
3
) does not require a trace element and it
also requires a significantly lower amount of nitrogen (14:1 C:N stoichiometric ratio).
The role of vitamin B
6
in the marine environment is also not as clear as that of vitamin
B
12
as auxotrophs for B
6
have not yet been identified. Further studies will be needed to
determine the uptake and production of this dissolved vitamin by marine phytoplankton
and bacteria and its role in the marine environment.

40

Vitamin B
7
Vitamin B
7
is well established as a required growth factor for many phytoplankton
species (Carlucci and Bowes, 1970b; Croft et al., 2006; Provasoli and Carlucci, 1974)
and this vitamin showed a variable distribution with a strong coastal influence with the
highest values in both transects being found at the coastal stations (11 and 12). It is
possible that this distribution is linked to nutrient availability highlighted by the overall
trend of higher concentration of vitamin B
7
along the more nutrient rich upper transect. It
is also possible that some leaching from the coastal sediments could be occurring at
stations 11 and 12. Another interesting feature was the peak of vitamin B
7
in the oxygen
minimum zone (OMZ) of station 11 which was not seen at for other vitamins or
methionine, and suggests that microorganisms in low oxygen environments may be
responsible for the high levels of dissolved B
7
either through excretion or cell lysis.
Our data further highlight the importance of the environmental availability of
vitamin B
7
by showing that phytoplankton in this region are potentially B
7
limited due to
an increase in chlorophyll with B
7
additions at station 7. The B
7
addition appeared to
specifically stimulate the growth of haptophytes which have not previously been
identified as B
7
auxotrophs in culture studies (Croft et al., 2006). Based on this finding,
we suggest further research into potential B
7
auxotrophy in haptophytes as it has been
previously suggested that vitamin B
7
is only required by phytoplankton that have
complex plastids (Croft et al., 2006) which are not found in haptophytes.

41

Summary and Conclusions

Overall, vitamins B
12
, B
6
, B
7
, and methionine were most abundant in the upper
500m with the highest concentrations being found at mid-depth in the euphotic zone
(Figures 1-9, 1-12, and 1-15) indicating a possible association with photosynthesis and
respiration processes. This trend was also found in a recent study of vitamin
concentrations in the California-Baja California coast (Sañudo-Wilhelmy et al., 2012). As
it has been suggested that phytoplankton excrete excess organic compounds at night
(Bjornsen, 1988), it is possible this production of vitamins may be associated with diel
photosynthetic activity. We suggest future studies correlating bacterial activity and
photosynthesis rates to vitamin and methionine concentrations to further explore this
possibility. While vitamin B
12
is only known to be produced by bacteria (including
cyanobacteria), it is possible that eukaryotic phytoplankton could produce other vitamins
such as B
6
and B
7
(Carlucci and Bowes, 1970a). The source of dissolved vitamins in the
ocean could therefore be either be directly associated with the activity of photosynthetic
organisms (i.e., cyanobacteria and eukaryotic phytoplankton) and/or indirectly associated
with secondary heterotrophic production.
Compared to vitamins measured in the California-Baja California coast, vitamins
in the ETSP were lower in concentration. In the California-Baja California region,
vitamin B
12
ranged from 30pM to undetectable, and B
6
and B
7
ranged from undetectable
to ~500pM (Sañudo-Wilhelmy et al., 2012). In the ETSP vitamin B
12
ranged from 6.99
pM to undetectable, B
6
ranged from 8.86 pM to undetectable, and B
7
ranged from 45 pM
to undetectable. These lower values are consistent with the oligotrophic conditions of
42

portions of the ETSP cruise track, in contrast to the nutrient enriched coastal ocean along
the northeast Pacific margin (Sañudo-Wilhelmy et al., 2012). Another notable difference
was that their geographical distribution was linked to water mass origin along the
California-Baja California coast but not in the ETSP. It is possible that the water masses
(such as the AIW and PDW) in the ETSP that contain growth factors (such as high levels
of nitrogen and phosphorous) required for plankton to synthesize vitamins were not being
sufficiently mixed into the upper 500m where vitamin excretion appears to primarily
occur, which is not the case in areas of the California-Baja California coastal margin
(Sañudo-Wilhelmy et al., 2012). One similarity between the two studies is that in both
locations, there were large areas where vitamins, such as vitamin B
12
, were undetectable.
It is possible that the community composition of microorganisms in these low vitamin
areas may be biased towards organisms that are able to synthesize vitamins de novo and
organisms that have mixotrophic lifestyles (and thus get their vitamins from smaller
prey/particles).
Therefore, while water masses and nutrient distributions do seem to impact the
distribution of some B-vitamins in some regions such as the California-Baja California
and the Long Island coasts (Gobler et al., 2007; Sañudo-Wilhelmy et al., 2012), our
results suggest that these trends do not hold for oligotrophic open ocean environments in
the ETSP. Our analysis also showed that the vitamin distributions were also independent
of basic microbial composition determined using flow cytometry. Therefore, the
biological activity or taxonomic composition of phytoplankton and bacteria in these
environments may be more important to understanding the cycling of B-vitamins in the
open ocean.
43

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47

CHAPTER TWO: THE IMPACT OF THIAMIN ON NITROGEN FIXATION BY
UNICELLULAR MARINE CYANOBACTERIA

Abstract
While organic cofactors such as B-vitamins are known to stimulate the growth of
many auxotrophic marine phytoplankton, their role in nitrogen fixation has not been
addressed. In this study, thiamin was selected for analysis due to putative thiamin
auxotrophy in uncultured UCYN-A diazotrophs as identified through genomic analysis.
The study site was selected due to the previously reported high abundances of these
organisms in the subtropical North Pacific. In order to investigate the impact of thiamin
(vitamin B
1
) on nitrogen fixation, vitamin B
1
was added to
15
N
2
uptake experiments
performed in the subtropical North Pacific. In addition to the field experiments, the effect
of thiamin amendments on nitrogen fixation was further substantiated in laboratory
cultures of two marine diazotrophs from different cyanobacteria groups: an identified
marine B
1
-auxotroph (Cyanothece 8902) and a B
1
-synthesizer (Crocosphaera WH0003).
In order to determine background levels of thiamin in the field location, dissolved
concentrations were measured during a diel cycle. Overall, nitrogen fixation was
significantly increased by the addition of thiamin in the field and in laboratory cultures.
Thiamin additions increased nitrogen fixation over the controls by 46% in the field and
by 127% in unialgal cultures of the auxotroph Cyanothece 8902, while no significant
change in nitrogen fixation was observed in the prototroph Crocosphaera WH0003.
Dissolved ambient thiamin concentrations were variable with depth, and concentrations
differed 233% to 1910% between day and night measurements at the same location.
48

Therefore, thiamin availability is another previously unrecognized factor which could
potentially influenced nitrogen fixation rates in the world ocean.

Introduction
Unicellular diazotrophs have recently been shown to be important contributors for
global nitrogen fixation budgets and are estimated to support significant amounts of new
production in the oligotrophic ocean (Montoya et al., 2004; Sohm et al., 2011; Zehr et al.,
2007). These open ocean diazotrophs include cyanobacterial groups UCYN-A, UCYN-B,
and UCYN-C, and have a widespread distribution in the world ocean (Falcon et al., 2002;
Zehr, 2011; Zehr et al., 2007). The UCYN-A group alone has been shown to occur with
up to 2.2 x 10
6
nifH gene copies per liter in the Pacific Ocean (Moisander et al., 2010).
While the essential nutrients P and Fe have been implicated as controllers of
nitrogen fixation in oceanic diazotrophs (Chappell et al., 2012; Mills et al., 2004;
Monteiro et al., 2011; Sañudo-Wilhelmy et al., 2001), the streamlined genomes of some
unicellular nitrogen fixers suggest that organic growth factors could also play a role in
their growth and nitrogen fixation rates (Karl, 2002; Tripp et al., 2010). Specifically, our
analysis of genomic data suggests that UCYN-A is a thiamin (B
1
) auxotroph. Thiamin is
an essential cofactor for the function of a variety of enzymes including transkelotase,
pyruvate oxidase, pyruvate decarboxylase, and deoxyxylulose 5-phosphate synthase
(Frank et al., 2007). Specific to nitrogen fixation, pyruvate-ferredoxin oxidoreductase, an
enzyme crucial for electron transfer to nitrogenase, requires thiamin (Bothe et al., 2010;
Brostedt and Nordlund, 1991). As such, this auxotrophy is expected to impact the
quantity and timing of nitrogen fixation by these important marine organisms.
49

In this study, we combine genomic analyses with field measurements and
shipboard and laboratory thiamin amendments to study the effect of this vitamin on
nitrogen fixation in the subtropical North Pacific. Laboratory cultures were performed
with a representative of UCYN-B (Crocosphaera WH0003) identified as being
prototrophic for thiamin and a representative of UCYN-C (Cyanothece 8902) identified
here as a B
1
auxotroph (UCYN-A is not available in culture). Day and night depth-
profiles of dissolved thiamin, measured at the field location where the amendments took
place, were used to establish the background levels and diel variability of this vitamin in
the tropical North Pacific during our experiments. The effect of diel changes in B
1

availability will help us to constrain the effect of this vitamin on rates of nitrogen
fixation, a process which also has a strong diel cycle (Church et al., 2005b; Gruber and
Sarmiento, 1997).

Methods

Thiamin Auxotrophy Conditions and Analysis
Genomic analysis of thiamin auxotrophy was performed using known genes with
IMG BLAST (e-value cutoff of < 1x10
-2
) and NCBI BLASTP. UCYN-A and
Crocosphaera WH0003 were analyzed for the presence of genes for the synthesis of the
pyrimidine moiety and the thiazole moiety of thiamin as defined according to Rodinov et
al., (2002). Gene abbreviations, accession numbers, and protein sequences used for
BLAST searches are presented in table 2-1.

50

Table 2-1: Thiamin biosynthesis genes used for auxotrophy identification.
Gene Abbreviation Accession number Protein sequence
ThiC ZP_00514294 MRTQWVAKRRGQSNVSQMHYARQGLITEEMDYV
AKREGLPPELIREEVARGRMIIPANINHPNLEPMCIGI
ASKCKVNANIGASPNSSDLDEEVAKLNLAVKYGAD
TVMDLSTGGGDLDTIRSAIIKASSVPIGTVPIYQAME
SVHGNMDKLCADDFLHIIEKHAQQGVDYMTIHAGI
LIEHLPLVRSRLTGIVSRGGGIIARWMLHHHKQNPL
YTHFDDIIEIFKKYDVSFSLGDSLRPGCTHDASDEAQ
LAELKTLGQLTRRAWEHDLQVMVEGPGHVPMDQI
EFNVRKQMEECSEAPFYVLGPLVTDIAPGYDHITSAI
GAAMAGWYGTAMLCYVTPKEHLGLPDAEDVRNG
LIAYKIAAHAADIARHRPGARDRDDQLSEARYNFD
WNRQFELSLDPERAKEYHDETLPADIYKTAEFCSMC
GPKFCPMQTKVDADALTELEKFLAEKDREKVAK
ThiD ZP_00519091 MGFSNIALTIAGSDSGGGAGIQADLKTFAFHCVHGT
SAITCITAQNTLGVNQVKKINSDLIKAQIEAIVTDINI
KAVKTGMLFDEEIIIAVSQQIKQWKLSQLVVDPVMV
SRTGVKLIDEGAIASLKTNLIPQALIVTPNRYEAQILS
NLSIFSLEDMKKAAQIIYNLGAKFVLIKGGGMKDNL
QGVDVWFDGKNCEVLTTETIKTKHTHGTGCTLSAA
ITANLALGKDPFIAVKQAKDYVTNALKYSLEIGKGT
GPVGHFFPLLKP
ThiF ZP_00516396 MNFTPTERERYSRQIQLPGFGEEGQKRLKDSTAVVT
GVGGLGGTVALYLAVAGVGKIILVRGGDLRLDDLN
RQILMTNSWVGQPRVYKARETLLNINPDIEVEAVSE
FVTAENIDALVQQANIAFDCAFDFKERHLLNQACVR
WGVPMVEAAMSGMDAYLTTVIPGETPCLSCIFPETP
EWDRWGFGVLGAVSGSLACLAALEGIKLLTGLGEP
LTGQLLTMDLGTATFAKRRPYHDPNCPVCGNFTKQ
RLSSYLPKQEELKIKK
ThiS ZP_00516114 MNTSEIVKLQVNGEEKTCSSGNNLPQFLVAMGLNP
RLIAVEYNGEILHRQYWENTILKTGDRLEIVTIVG
GG
ThiO/ThiG ZP_00513674 MNATNDIIIIGGGIIGMAIAVDLKLRGASVTVCNRNF
PQTASVAAAGMLAPHAEELPPGPLLDLCLKSRWLY
PEWVRKLQDLTGLDLGYNPSGILAPVYDLPGVDVR
NHNQSQWLDKTAIRLCQTGLGDDVVGGWWYPEDG
QVDNRQVMQALRQAAQQLGVNLRDGVTIQTLQQK
QGQVTSILTNQGELSGNTYIIANGSWASQILPLPVRPI
KGQMLAVKMPHTPNEPYPLQRVLYGPQTYLVPRQN
GRLIIGATSEDVGWTPHNTPQGMETLMKRAIRLYPD
IANWEIEEFWWGYRPGTPDELPILGHYGCDNLILAT
GHYRNGILLAPATASLIADLVINQQTDPLLENFKGD
RFHTQPSPPPAPMTSFNVYPVPTTINGNNGNSSLSSP
DELIIAGRKFRSRLMTGTGKYPTISIMQESVAASECQI
VTVAVRRVQTKAPGHEGLAEALDWDKIWMLPNTA
GCQTAEEAIRVARLGREMARLLGQEDNNFVKLEVIP
DSKYLLPDPIGTLEAAETLVKEGFAVLPYINADPILA
KRLEEVGCATVMPLGSPIGSGQGIRTQANIEIIIEEAN
IPVVVDAGIGTPSEASQAMEMGADAVLINSAIALAK
NPVLMARAMGMATVAGRLAYLSGRIPVKDYAIASS
PLTGTVV

51

In order to complement the genomic identification of auxotrophy, triplicate
unialgal cultures of Cyanothece 8902 were grown in modified YBC-II as described in
Chen et al., (1996) with the exception of the vitamin mixture. YBC-II media was
modified with the following treatments: 1) 1nM B
1
and 10 nM B
12
and 2) 10 nM B
12
and
no B
1
. Cultures were inoculated with 1ml of culture in exponential growth phase in 45 ml
polycarbonate culture tubes and grown at 150 mol photons/ m
2
/ sec
-1
on a 12:12
light:dark cycle at 25˚C in batch culture. Culture growth was measured using chlorophyll
a fluorescence measured with a Turner 10-AU fluorometer.

Field Incubations
Field amendment experiments were conducted in the North Pacific off Hawaii
(20’30.053˚N, 157’40.400˚W) in J uly 2010 under trace metal clean conditions.
Incubations were conducted with water collected from a depth of approximately 10 m
and prefiltered through 200 m nylon (Nitex) screening to reduce grazer abundance.
Sample bottles (2L polycarbonate) were filled to overflowing and sealed with a septum
cap. Care was taken to ensure no air bubbles were present in the bottles. Samples were
injected with 0.5 ml
15
N
2
gas using a gas tight syringe as described in Montoya et al.,
1996). Thiamin spikes were passed through Chelex as described in Price et al., (1988/89)
to remove any trace metal contaminants. Samples were incubated in a deck board
incubator at light and temperature levels set up to mimic in-situ conditions. Samples were
incubated for 48 hours; including 500 pM B
1
spike treatments and control treatments with
no amendments. After the incubation time, all of the samples were filtered onto
precombusted GF/F filters and stored at -20˚C until analyzed. Samples were then dried at
60˚C, wrapped in Heraeus CHN cups, and pelletized. Samples were analyzed for
15
N

52

signatures at the UC-Davis Stable Isotope Facility. Outliers in biological triplicates were
identified using R version 2.9.0 (http://www.r-project.org/, accessed May 2012).

Culture Nitrogen Fixation Conditions and Analysis
As UCYN-A is not available in culture, Cyanothece 8902 (a group C
representative that is the most closely related strain to group A cyanobacteria available in
culture; Taniuchi et al., 2012) was used for culture nitrogen fixation analysis. The more
distantly related group B diazotroph Crocosphaera WH0003 was also used as a
putatively prototrophic control. Unialgal stock cultures of Crocosphaera WH0003 and
Cyanothece 8902 were incubated at 28°C with a light:dark cycle of 12:12 and an incident
photon flux density of 100 µmol photons m
-2
s
-1
. All cultures were grown in 0.2 µm
filtered and autoclaved artificial seawater enriched with AQUIL nutrients (Price et al.,
1988/89) with no added nitrogen sources. Cultures were incubated in triplicate in 250 ml
autoclaved-polycarbonate bottles containing sterilized artificial seawater under the same
growth conditions as the stock cultures, but either with or without vitamin B
1
added (the
final concentration of B
1
in the medium was 296 nM). A 5-ml aliquot of stock culture
was transferred to 200-ml B
1
-free medium in order to minimize the carryover of B
1
from
the stock cultures. Experiments used identical semi-continuous culturing methods with
each species (Fu et al., 2008), and all cultures were acclimated to their respective growth
medium for at least 14 days to ensure long-term acclimation. Cultures were considered
B
1
-limited when the growth rates of B
1
-free cultures were significantly lower than those
of the B
1
-replete cultures. To determine whether the addition of B
1
will stimulate N
2

fixation of B
1
-limited Crocosphaera and Cyanothece, 1nM B
1
was added to B
1
-limited
cultures. Two experimental conditions were used: 1) 1nM B
1
and 2) B
1
free controls.
53

Nitrogen fixation was measured by acetylene reduction after a 48 hour incubation period
(Capone, 1993; Fu et al., 2008).

Dissolved Thiamin Analysis
Depth profiles within the photic zone (from surface to 150m) from B
1
analysis
were collected during a night/day cycle (12am/12pm) from the hydrocast in amber bottles
and immediately filtered through 0.2 m Supor® membrane filters into methanol cleaned
1L amber HDPE bottles and frozen at -20˚C for later analysis in the laboratory. Vitamins
were extracted from seawater samples by solid phase extraction using C
18
resin and
analyzed by LC-MS as described in Sañudo-Wilhelmy et al. (2012).

Results

Culture Auxotrophy Analysis
Genomic analysis identified UCYN-A and Cyanothece 8902 as thiamin
auxotrophs due to the presence of thiamin requiring enzymes (e.g. transkelotase) and the
lack of key genes required for the biosynthesis of the vitamin. Specifically, UCYN-A was
found to lack the genes required for synthesizing the pyrimidine moiety of thiamin (thiC
and thiD; table 2-2). In contrast, Crocosphaera strain WH0003 was found to have all
required thiamin biosynthesis genes (as described in Rodionov et al., 2002).
Table 2-2: Thiamin biosynthesis genes in select unicellular diazotrophic unicellular cyanobacteria.
pyrimidine moiety thiazole moiety
ThiC ThiD ThiF ThiS ThiO/ThiG
UCYN-A absent absent X X X
Crocosphera WH0003 X X X X X
54

Cultures of Cyanothece 8902 were unable to sustain growth without the addition
of vitamin B
1
and were determined to be auxotrophs for this compound. After 69 hours,
cultures reached a concentration of 416 ± 10 g/ml Chla when grown in media
containing B
1
, and only 0.5 ± 0.2 g/ml Chla in media lacking B
1
(Figure 2-1).
Hours
0 20 40 60
g/ml Chla (with thiamin)
0
100
200
300
400
g/ml Chla (without thiamin)
0
2
4
6
8
10

Figure 2-1: Growth of Cyanothece 8902 with thiamin (black) and without thiamin (red).

Impact of Thiamin on Nitrogen Fixation
In the North Pacific incubation experiments, nitrogen fixation rates for control
incubations were 1.12 ± 0.01 mol N fixed/l/day, compared to 1.63 ± 0.22 mol N
fixed/l/day for incubations with thiamin additions. This represents an average increase of
46% in nitrogen fixation rates with the addition of the B-vitamin with the control values
being significantly lower than the mean of the thiamin addition treatments (p-value =
0.01) (Figure 2-2).
55

Control Thiamin
mol N fixed
-1
day
-1
0.0
0.5
1.0
1.5
2.0

Figure 2-2: Nitrogen fixation values for control and thiamin addition incubations calculated as mol N
fixed/liter/day in 48 hour
15
N
2
field incubations. Error bars are standard error of biological replicates.

These values are within the range of nitrogen fixation rates previously reported for the
subtropical North Pacific (from 0.38 mol N fixed/l/day to 1.68 mol N fixed/l/day;
(Grabowski et al., 2008), with the nitrogen fixation rate in the thiamin addition being at
the high end of this range. While the overall nitrogen fixation rates are expected to be
underestimates according to a recent study on the solubility of
15
N
2
in incubation analyses
(Mohr et al., 2010), the overall trends are expected to remain unaffected especially due to
the 48 hour incubation time of our amendments.
Culture nitrogen fixation experiments performed with the B
1
auxotroph
Cyanothece 8902 showed an average increase in nitrogen fixation of 127% with the
addition of thiamin over the controls (2.5 ± 0.40 fmol/cell/hr thiamin treatment vs. 1.1 ±
0.1 fmol/cell/hr control; p-value = 0.003) (Figure 2-3). Nitrogen fixation rates were
essentially the same with or without the addition of thiamin (2.6 ± 0.4 pmol/cell/hr vs. 2.9
± 0.1 fmol/cell/hr; p-value = 0.28) in Crocosphaera WH0003 cultures (Figure 2-3).
56

fmol N fixed cell
-1
hour
-1
(Cyanothece 8902)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Cyanothece 8902 (Control)
Cyanothece 8902 (Thiamin)
Crocosphaera WH0003 (Control)
Crocosphera WH0003 (Thiamin)
fmol N fixed cell
-1
hour
-1
(Crocosphaera WH0003)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Figure 2-3: Nitrogen fixation rates in fmol/cell/hour in Cyanothece 8902 and Crocosphaera WH0003 with
control (black) and thiamin (grey) treatments. Error bars are standard deviation of biological replicates.

Dissolved Thiamin Analysis
Dissolved thiamin values measured in upper 150 meters at the station off Hawaii
ranged from 0.35 to 10 pM (Figure 2-4). A comparison between night and day depth
profiles reveals an inverse relationship between the measurements (Figure 2-4, r
2
= 0.86).
Below the mixed layer depth (40 - 60m) where levels of photosynthetically active
radiation (PAR) are low, B
1
appears to accumulate during the day and be drawn down at
night. At intermediate depths within the mixed layer, high values of thiamin are observed
at night (75m – 9.7 pM) with a decrease observed during the day (75m – 0.91 pM).
Surface values follow a different trend with similar values occurring during night (4 pM)
and day (6 pM).

57

Thiamin (pM)
0 2 4 6 8 10 12
Depth (m)
0
20
40
60
80
100
120
140
PAR (µE/m
2
/s)
0 10 20 30 40 50
Temperature (ºC)
19 20 21 22 23 24 25 26
Thiamin (pM)
0 2 4 6 8 10 12
0
20
40
60
80
100
120
140
PAR (µE/m
2
/s)
0 10 20 30 40 50
Temperature (ºC)
19 20 21 22 23 24 25 26
(A) (B)

Figure 2-4: Day (A) and Night (B) profiles of thiamin (black), photosynthetically active
radiation (PAR), and temperature (blue). Profiles were taken at 24:00 (Day) and 12:00
(Night).

Discussion
While controls on nitrogen fixation have previously been attributed largely to the
availability of Fe and P (Mills et al., 2004; Monteiro et al., 2011; Sañudo-Wilhelmy et al.,
2001), or more recently CO
2
(Fu et al., 2008; Hutchins et al., 2007), our results
demonstrate that increased thiamin availability also causes an increase in the rate of
nitrogen fixation in field incubations, as well as in laboratory experiments. The increase
observed in the station off Hawaii in the B
1
incubation is attributed to auxotrophic
organisms, specifically the abundant UCYN group A unicellular diazotrophs which our
genomic analyses suggest is a thiamin auxotroph. In culture, a thiamin auxotroph
(Cyanothece 8902) also shows a dramatic 127% increase in nitrogen fixation with the
addition of B
1
. These results suggest that thiamin auxotrophs may be found in a variety of
58

unicellular diazatroph groups, and that the availability of thiamin in the marine
environment can influence the rate of nitrogen fixation by these organisms.
The importance of thiamin availability for auxotrophic diazotrophs appears to be
complicated by the environmental variability observed in the depth profiles measured in
this study. Those profiles showed an inverse relationship between night and day
concentrations varying with depth. For example, B
1
concentrations at the surface (5m)
and at the bottom of the euphotic zone (150m) were similar between night and day, while
an inverse trend was observed at intermediate depths. This strong diel trend is similar to
the diel pattern seen for other organic compounds in the ocean such as free amino acids
(Carlucci et al., 1984), where the amino acid flux was coupled to primary production. It
has previously been suggested (Bjornsen, 1988) that phytoplankton could potentially be
exuding organic compounds at night as a result of passive diffusion of an overflow of
organic material produced during photosynthesis. This would be consistent with the high
levels of thiamin we detected at night.
These diel changes in thiamin concentrations may influence the ability of
auxotrophic organisms to perform nitrogen fixation at certain times and depths, especially
considering that many diazotrophs have diurnal nitrogen fixation cycles with group A
organisms showing nifH transcription during the day and group B and group C organisms
fixing nitrogen primarily at night (Church et al., 2005b; Needoba et al., 2007). Therefore,
the diel cycle of thiamine observed at our sampling location could be reflecting the fact
that some auxotrophic organisms may be taking up during the day the excreted thiamin
produced at night, resulting in the low daytime levels. Consistent with that hypothesis,
Group A cyanobacteria have previously been reported (Church et al., 2005a) to occur
59

primarily in the high light-low nutrient surface waters where we measured relatively high
levels of thiamin at night (6 – 10 pM at 5 – 20 m depth). In addition, some auxotrophic
organisms could also be associated with night (10 pM – 75m) and day (7 pM – 100 &
150m) maxima at deeper depths where relatively high abundances of cyanobacteria and
genes related to thiamin metabolism (at 130m) have previously been reported (DeLong et
al., 2006).
While half-saturation constants for thiamin uptake in cyanobacteria have not been
reported, the observed concentrations are at the low end of reported half-saturation
constants for eukaryotic phytoplankton (5.94 – 184 pM; Tang et al., 2010). If
cyanobacteria have similar affinities for thiamine as eukaryotes, this could indicate that in
this region they may be limited by the availability of thiamin. The fact that thiamin
additions increase nitrogen fixation suggests that that is the case. It is also likely that
thiamin concentrations could impact nitrogen fixation in other regions of the world ocean
with high abundances of group A cyanobacteria, such as the Arabian Sea and the eastern
Atlantic (Taniuchi et al., 2012; Zehr, 2011). Although less is known about the
distributions and abundance of group C cyanobacteria (Taniuchi et al., 2012), our results
suggest that their nitrogen fixation rates too may be potentially limited by thiamin. The
fact that group B cyanobacteria like Crocosphaera do not appear to require exogenous
thiamin sources suggests the intriguing possibility that this vitamin could affect the
relative competitive dominance and community structure of these three common groups
of diazotrophs. Further research is needed to identify the specific biological production
and uptake that are likely causing the diel variations in thiamin profiles, in order to gain
further understanding on the impact of this variation on auxotrophic marine organisms.
60

Overall, this study shows that organic compounds, such as some B-vitamins, could be
important controllers of biological processes such as nitrogen fixation in the marine
environment and highlights the importance of further research in this area.

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63

CHAPTER THREE: STATUS OF METAL CONTAMINATION IN SURFACE
WATERS OF THE COASTAL OCEAN OFF LOS ANGELES, CALIFORNIA
SINCE THE IMPLEMENTATION OF THE CLEAN WATER ACT

Abstract
In order to establish the status of metal contamination in surface waters in the
coastal ocean off Los Angeles, California, we determined their dissolved and particulate
pools and compared them with levels reported in the 1970s prior the implementation of
the Clean Water Act. These measurements revealed a significant reduction in particulate
toxic metal concentrations in the last 33 years with decreases of ∼100-fold for Pb and
∼400-fold for Cu and Cd. Despite these reductions, the source of particulate metals
appears to be primarily anthropogenic as enrichment factors were orders of magnitude
above what is considered background crustal levels. Overall, dissolved trace metal
concentrations in the Los Angeles coastal waters were remarkably low with values in the
same range as those measured in a pristine coastal environment off Mexico’s Baja
California peninsula. In order to estimate the impact of metal contamination on regional
phytoplankton, the internalization rate of trace metals in a locally isolated phytoplankton
model organism (Synechococcus sp. CC9311) was also determined showing a rapid
internalization (in the order of a few hours) for many trace metals (e.g., Ag, Cd, Cu, Pb)
suggesting that those metals could potentially be incorporated into the local food webs.

64

Introduction
The Southern California Bight (SCB) is a densely populated and industrialized
area subject to high levels of anthropogenic inputs from wastewater treatment plants,
urban and agricultural runoff, oil and gas production, vessel activities, and hazardous
material spills (Schiff et al., 2000). Nowhere is this more evident than in the coastal
ocean off Los Angeles, California. Los Angeles County houses an estimated 10 million
inhabitants (The County of Los Angeles Annual Report, 2009-2010) that, together with
other SCB counties, generate more than 4.7 billion gallons of treated effluent water per
day (Lyon and Stein, 2009). This effluent water is discharged into the coastal ocean by
nineteen municipal wastewater treatment plants serving the Los Angeles area, including
the large Hyperion Treatment Plant (HTP) (City of Los Angeles) and the Joint Water
Pollution Plant (JWPCP) (Los Angeles County Sanitation Districts) (Lyon and Stein,
2009). This effluent water is discharged five miles offshore at a depth of 60 m (Schiff and
Bay, 2003). While regulation through the Clean Water Act led to a large reduction in the
input of pollutants into the SCB beginning with its implementation in 1972 (Sañudo-
Wilhelmy et al., 2004), effluent discharge from those wastewater treatment plants
continues to discharge several metric tons of toxic metals such as Ag, Cu, Ni, Pb, and Zn
into the Bight every year (Galloway, 1979; Lyon and Stein, 2009; Schiff et al., 2007).
Current monitoring programs within the SCB have primarily focused on determining
temporal and spatial changes in metal contamination in sediments and biota (Brown et
al., 1987; Huh, 1996; Phillips, 2007; Santschi et al., 2001), and, therefore, current data on
the concentrations of water-column particulate and dissolved metals in the marine
environment off Los Angeles are very limited. There is also no information about metal
65

accumulation within local phytoplankton species even though several studies have shown
that environmentally relevant trace metals, including Cu, Ni, Pb, and Cd are readily
internalized by phytoplankton (Cullen et al., 1999; Dupont et al., 2008; Quigg et al.,
2006; Sanchez-Marin et al., 2010; Sunda and Huntsman, 2000). In this study, we assayed
the impact of the Clean Water act on toxic trace metals in surface waters of the SCB off
Los Angeles by determining current levels of particulate and dissolved metals and
comparing these levels to measurements done in the same locations in the early 1970s by
Bruland and Franks (1978). In addition, enrichment factors calculated for particulate
trace metals and a comparison of dissolved trace metal levels measured off Los Angeles
with those measured in a pristine environment off Punta Banda, Baja California, Mexico
(33° N, 117° W) and elsewhere in the SCB in 1989 were used to evaluate the current
status of metal contamination within this area of the Bight. In order to more fully
understand the ecological impact of dissolved metals in these coastal waters, we also
determined trace metal internalization in the cyanobacteria Synechococcus sp. CC9311 in
an attempt to establish the potential for biological uptake by local phytoplankton.

Materials and Methods

Sample Collection and Study Site
Surface water samples were collected in the SCB off Los Angeles in February and
September of 2009 (Figure 3-1).
66

JWPCP Plant
Hyperion Treatment Plant
San Gabriel
River
4001
4004
3904
3906
3802
3804
3806
3704
3606
3505
3504
3304
4006
3306
3204
3206
3104
3106
3056
3003
2903
3006
2906
2802
2805
2706
2602
2604
2606
2501
2506
2503
Los Angeles
Palos Verdes
Peninsula
Point Dume
Los Angeles
River

Figure 3-1: Map of SCB sampling sites with potential sources of metal input indicated with arrows.
Sampling locations are identified using the station numbers of the Los Angeles County Sanitation District
and the City of Los Angeles Sanitation District monitoring programs. Map was generated using Ocean Data
View (Schlitzer, 2011).

February samples were collected in collaboration with the Los Angeles County Sanitation
District and the City of Los Angeles Sanitation District, and September samples were
collected in collaboration with the USC Wrigley Institute for Environmental Studies.
Station numbers correspond to environmental monitoring stations of these agencies.
Detailed descriptions of the physical setting of the sampling area have been previously
reported.19,20 All samples were collected using trace metal clean techniques at a depth
of approximately 1−2 m from the surface and refrigerated until filtration (<12 h later).
Samples for dissolved trace metal analyses were also collected biweekly in Punta Banda,
Mexico from 2004 to 2005.

67

Particulate and Dissolved Metal Analyses
Refrigerated samples were filtered (1.5 to 2 L) through acid-washed and
preweighed 0.45 μm polycarbonate filters to distinguish between particulate (>0.45 μm)
and dissolved (<0.45 μm) trace metals. Filtration was performed in a class-100 clean
room, and samples were handled using trace metal clean techniques. Dissolved samples
were acidified using Optima grade hydrochloric acid to a pH <2 and stored for at least
one month prior to preconcentration by organic extractions with the APDC/DDDC ligand
technique described in Bruland et al. (1985). Particulate samples were dried to determine
particulate dry weight and analyzed for refractory and labile metal concentrations as
described in Bruland and Franks (1978). This type of sequential leaching is commonly
used to determine which particulate metals are likely to be readily desorbed from
suspended particles (labile) from those more strongly bound (refractory) (Bruland and
Franks, 1978). The labile pool was determined by placing the filters in an acetic acid
leach (20% Optima grade acetic acid for two hours). After the labile leach, the refractory
metal pool was then obtained by boiling the filters for 45 min in acid-washed Teflon
digestion bombs with Optima grade HF, HCl, and HNO
3
. Total particulate trace metal
concentrations are reported as the labile + refractory trace metal pools. Trace metal levels
in all the particulate and dissolved pools were quantified by ICPMS using external
calibration curves and an internal indium standard.

Phytoplankton Metal Internalization Experiments
Axenic cultures of Synechococcus sp. CC9311, a strain isolated from the
California current, were grown in filtered, amended SCB seawater (collected using trace
68

metal clean techniques from the San Pedro Oceanographic Times Series station (SPOTS)
during November 2009) in acid washed polycarbonate containers at 18 °C at a 12/12 light
cycle of 100 μmol photons m−2 s−1. The SCB media was microwave sterilized (Keller et
al., 1988), pH adjusted to 8.0−8.2 with sodium hydroxide, and amended with N and P
(final concentrations: 8.0 × 10−4 M Optima grade nitric acid and 5.0 × 10−5 M
phosphoric acid).
Cultures were acclimated to the modified SCB seawater media for 3 transfers
prior to transfer to 2 L experimental vessels. The purity of the cultures was confirmed at
each time point via examination of DAPI (4′,6 -diamidino-2-phenylindole) stained
aliquots using a Zeiss Axiostar epifluorescent microscope and subsample addition to
Marine Purity Broth (Bertilsson et al., 2003). Cell growth was estimated through
microscopic examination and flow cytometry at each time sampling point. For flow
cytometry (FC), samples were fixed with a final concentration of 0.1% formalin prior to
analysis and run along with an internal standard of BD on a FACSCalibur Flow
Cytometer. FC results were analyzed using the CellQuest software (BD Biosciences).
Trace metals internalization experiments were performed in 2 L acid washed
polycarbonate bottles that were amended with bioactive (nutrient and toxic) trace metals
at concentrations approximately 5× the dissolved concentrations found at neareffluent
discharge stations in the Palos Verdes area during February 2009 (0.5 nM Al, 15 nM Ni,
10 nM Cu, 0.05 nM Ag, 1 nM Cd, 0.3 nM Pb, 595 nM Mo, 1 nM Co, 15 nM Zn, and
15nM Fe). Amended and control cultures were filtered down at 0, 3, 6, 12, 24, and 48 h
after trace metal additions. At each time point, 50 mL aliquots were filtered onto acid-
washed 0.45 μm polycarbonate filters for total metal and intracellular metal
69

concentrations. The intracellular pool was determined using the oxalate wash procedure
(Hassler and Schoemann, 2009; Tovar-Sanchez et al., 2003). Trace metals were extracted
with heated acid digestions in sealed Teflon vessels containing Optima grade nitric and
hydrochloric acids. Trace metal analysis of digested solutions was performed by ICPMS
as described above. Trace metal detection limits and procedural blanks can be found in
the Supporting Information.

Results and Discussion

Spatial Distribution of Particulate Trace Metals
Actual metal concentrations in the dissolved, particulate and intracellular pools as
well as other ancillary parameters are presented in the Appendix A. Geographical
distribution of Zn, Fe, Co, and V (in our study area) were similar to those observed for
Cu and Pb (Figure A2 Appendix A). Spatial distribution of particulate Cu, Co, Fe, V, and
Zn suggest that point sources and stormwater runoff were likely contributors of
particulate metals to the coastal ocean off Los Angeles during our sampling. This is
indicated by high concentrations of these metals measured in the vicinity of the San
Gabriel River (7.7 nM Cu, 1.9 nM Co, 8.5 nM Fe, 23 nM V, and 19 nM Zn), the Los
Angeles River/Long Beach Port area (9.5 nM Cu, 2 nM Co, 8.2 nM Fe, 26 nM V, and 19
nM Zn), north of the HTP outfall (6 nM Cu, 610 pM Co, 3.2 nM Fe, 9.2 nM V, and 16
nM Zn), and near the JWPCP outfall at White Point (6.7 nM Cu, 1.6 nM Co, 8.3 nM Fe,
25 nM V, and 15 nM Zn). Particulate Ba levels were elevated in the White Point and Los
Angeles River/Port of Long Beach areas (mean 118 ± 6 nM) (Figure 2) suggesting a
70

potential contribution from vehicle emissions (Monaci and Bargagli, 1997) or from oil
contamination (Chow et al., 1978) to the ambient metal load in these regions. In contrast
to the other metals, particulate Cd showed a different distribution with levels being fairly
uniform throughout the sampling area with the highest levels (400−200 pM) observed off
the coast of Malibu, potentially due to differences in the point sources of particulate Cd
(Figure 3-2).
Cadmium [nM]: February 2009 (>0.45 m)
Copper [nM]: February 2009 (>0.45 m)
Lead [pM]: February 2009 (>0.45 m) Barium [nM]: February 2009 (>0.45 m)

Figure 3-2: Concentration gradient maps of particulate Cd, Cu, Ba, and Pb measured in February 2009 in
the SCB. The metal concentration range is indicated with the different colors. Maps were generated using
Ocean Data View (Schlitzer, 2011).

Although the high levels of metals near to effluent inputs are not totally
unexpected due to the large volume of water being discharged into the area, their
71

presence in areas up the coast from the HTP outfall suggests a potential
horizontal/vertical transport of effluent particles in the coastal regions of Santa Monica
Bay. The high levels of particulate Al detected near the San Gabriel River (1.2 nM) and
Los Angeles River (670 nM) suggest that the metals in those regions could be from
terrigenous sources. We cannot rule out other sources such as atmospheric deposition
which has been shown to be an important source of some particulate metals (e.g., Cu) to
the region (Sabin and Schiff, 2008). However, high concentrations of sewage-tracer
dissolved Ag (Sañudo-Wilhelmy and Flegal, 1992) (Figure 3-5) near the river locations
might suggest that the ultimate source of these riverine metals are from upriver
discharges from water reclamation plants, National Pollutant Discharge Elimination
Systems, power plants discharge, and/or storm drains (Ackerman et al., 2005; Foster,
2005).

Temporal Gradients in Particulate Trace Metals
A comparison between the levels of particulate trace metals measured in February
2009 samples and those measured in the same vicinity in February 1976 by Bruland and
Franks (1978) shows that the overall levels of particulate metals has been largely
reduced. Specifically, concentrations measured in samples collected near the JWPCP
outfall and HTP outfalls have declined ∼400-fold for Cd and Cu, ∼100-fold for Pb and
V, ∼50 fold for Ni, and ∼10-fold for Zn and Ba relative to 1976 (Figure 3-3A). Similar
reductions were observed when comparing the concentrations measured at the near-shore
stations (noneffluent discharge stations located anterior to the inner basin in 1976
(Bruland and Franks, 1978) and the 2009 samples closest to those stations (Figure S1
72

Supporting Information) between 1976 and 2009 ( ∼60-fold decrease in Cu, a ∼30-fold
decrease in Cd, Pb, and Zn, and a ∼6-fold decrease in Ni and V). This metal
concentration decline is consistent with reductions in mass discharges from the large
treatment plants into the SCB (from 1,184 × 109 L per year in 197632 to 1,402 × 109 L
per year in 2009). The exception to this temporal trend was Ba concentrations, which, on
average, were ∼1.5 times higher in 2009 at our near-shore stations, potentially due to
their inclusion in antifouling paints and association with processed gasoline (Chow et al.,
1978; Monaci and Bargagli, 1997; Turner, 2010).
73

Cd Zn Pb Ba Ni Cu V
Cd & Zn (ppm dry weight)
0
20
40
60
Ni & Cu (ppm dry weight)
0
20
40
60
Pb & Ba (ppm dry weight)
0
50
100
200
250
V (ppm dry weight)
0
50
100
800
1000
A
Log
10
([Metal Sample]/[Fe Sample])/([Metal Crust]/[Fe Crust])
1e-3 1e-2 1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5 1e+6
Co
Cu
Ni
V
Zn
Ba
Cd
Pb
B

Figure 3-3: (A) Particulate metal concentrations measured at the near-effluent outflow stations in February
1976 (black bars) and February 2009 (gray bars). The1976 values are mean values obtained at stations 443
(surface sample) and 361 (10 m off the bottom) (Bruland and Franks, 1978). The 2009 concentrations are
mean ± standard deviation from stations 2802, 2903, and 3504 (Figure 3-1). The location of the 2009
stations was selected based on their proximity to 1976 stations. Station coordinates are available in the
Supporting Information. (B) Box-plots of the enrichment factors (EF) for particulate metals calculated for
all February 2009 stations. The dashed line represents the EF 1 order of magnitude above what is
considered crustal levels (Schiff and Weisberg, 1999; Wedepohl, 1995).

The overall characteristics of the particulate trace metals in surface water samples
remained largely unchanged with the average percent labile particulate metals being
lower in 1976 but within the same range (97% labile Cd in 1976 vs 91% ± 20 labile Cd in
74

2009; 38% labile Cu in 1976 vs 52% ± 30 labile Cu in 2009; 61% labile Pb in 1976 vs
76% ± 26 labile Pb in 2009; 62% labile Zn in 1976 vs 54% ± 29 labile Zn in 2009; 64%
labile Ba in 1976 vs 42% ± 27 labile Ba in 2009; 67% labile V in 1976 vs 45% ± 27
labile V in 2009) with the exception of Ni which had a lower average percentage of labile
particulates in 2009 (57% labile Ni in 1976 vs 27% ± 25 labile Ni in 2009).
The reduction in particulate metal levels observed in the last 33 years in the
coastal ocean off Los Angeles is not due to improvements in sample collections and/or
analytical protocols as both sets of samples were collected and analyzed using similar
protocols. Furthermore, to reduce the seasonal and spatial variability, both sampling
campaigns took place in February (1976 and 2009) at the same locations or within the
vicinity of each other.

Potential Sources of Particulate Trace Metals
The source of particulate Cu, Ni, Zn, Ba, Cd, Pb, and Ba appears to be primarily
from anthropogenic sources. This is based on an enrichment factor analysis (EF) in which
metal concentrations are normalized using the equation [Metal]/[Fe]
sample
/[Metal]/[Fe]
crust

where [Metal]/[Fe]
sample
represent the concentration of the metal of interest and Fe in the
particulate surface water sample and [Metal]/[Fe]
crust
represent the average concentration
of the metal of interest and Fe in the crust (Schiff et al., 2000). EF analysis has been
shown to be a successful indicator of anthropogenic sources of metals in particulate
matter (Amin et al., 2009; Schiff and Weisberg, 1999; Selvaraj et al., 2010; Valdes et al.,
2005; Zhang and Liu, 2002).
75

In this analysis, enrichment factors were highest for Pb and decreased on average
as Pb > Cd > Ba > Zn > V > Ni > Cu >Fe > Co (Figure 3-3B). The most highly enriched
particulate metals were Pb and Cd, which had ratios of, on average, ∼4 and ∼2 orders of
magnitude above crustal levels, respectively. Both Cd and Pb have shown elevated
enrichment factors relative to other metals and have been therefore implicated as having
anthropogenic sources (Amin et al., 2009; Zhang and Liu, 2002). Cu, another particulate
trace metal of interest due to its known toxicity to picoplankton and association with
antifouling paint, (Debelius et al., 2009; Schiff et al., 2007; Turner, 2010) had enrichment
factors ∼1−2 orders of magnitude above crustal levels in stations near the Port of Long
Beach and the San Gabriel and Los Angeles rivers. The elevated enrichment factor of Ba
also suggests an anthropogenic source of this particulate metal, while the lower EFs for
the remaining metals indicates that these metals are likely to have primarily natural
sources. Further research will be required to definitively link the distribution of these
metals to specific sources and calculate realistic mass balance estimates for the SCB.

Distribution of Dissolved Trace Metals
Variations in water circulation patterns within the SCB and stormwater runoff and
sewage are likely to be major factors affecting the distribution of dissolved trace metals
in the Los Angeles area. The major point sources influencing metal levels in the February
2009 cruise appear to be the San Gabriel River as elevated levels of Ag (13 pM), Cu (5
nM), Cd (210 pM), and Pb (100 pM) were all detected near the river outflow (Figure 3-
4). Mean and median concentrations for dissolved metals for that cruise (mean ± standard
deviation/median) were Ag, 6.8 ± 3.6 pM/6.3 pM; Cu, 1.4 ± 0.9 nM/1.1 nM; Cd, 120 ±
76

31 pM/118 pM; and Pb, 43 ± 18 pM/37 pM. To a lesser extent, relatively high levels
( ∼12 pM) of sewage-tracer Ag (Sañudo-Wilhelmy and Flegal, 1992) were also measured
in the vicinity of the JWPCP and HTP effluent discharge outfalls (Figure 3-4). The
elevated levels of Ag in these areas (San Gabriel River outflow and near JWPCP and
HTP’s outfalls) suggest that some effluent discharge reached the surface waters of these
locations.
77

Cadmium [pM]: February 2009 (<0.45 m) Cadmium [pM]: September 2009 (<0.45 m)
Copper [nM]: February 2009 (<0.45 m)
Copper [nM]: September 2009 (<0.45 m)
Silver [pM]: February 2009 (<0.45 m) Silver [pM]: September 2009 (<0.45 m)

Figure 3-4: Concentration gradient maps of dissolved Ag, Cu, and Cd measured in February and
September 2009 in the SCB. The metal concentration range is indicated with the different colors. Maps
were generated using Ocean Data View (Schlitzer, 2011).

In contrast to the geographic patterns observed in February, relatively high
dissolved metal concentrations in September were observed at stations located north of
78

the Palos Verdes Peninsula (4 nM Ni; 49 nM V; 169 nM Mo; 172 pM Co; 7 pM Ag,) and
off of Point Dume (252 pM Cd; 12 nM Fe) (Figure 3-5 and Figure A-4 in Appendix A).
The mean and median dissolved concentrations of these metals in September 2009 (mean
± standard deviation/median) were Ni, 2.0 ± 0.5 nM/2.0 nM; V, 20 ± 6.8 nM/19 nM; Mo,
67 ± 22 nM/64 nM; Co, 66 ± 34 pM/55 pM; and Ag, 2.9 ± 1.3 pM/2.6 pM. Coastal
currents and upwelling events could potentially explain the seasonal variations observed
for some metals such as Cd, Co, Ni, Mo, V, and Fe that are strongly influenced by
circulation patterns, seasonal nutrient distributions, and biological activity (Dellwig et al.,
2007; Di Lorenzo, 2003; Mackey et al., 2002; Sañudo-Wilhelmy and Flegal, 1996; Wang
and Wilhelmy, 2009). As the February sampling was carried out during the rainy season
(2.15−3.65 in. of precipitation measured at Santa Monica and Palos Verdes during the
week of our cruise) (Western Regional Climate Center, 2011), differences in metal
concentrations and distributions are also likely to be related to variations in river inputs
(mean discharge from the San Gabriel River of 0.067 ft
3
/s in February 2009 vs 0.0 ft
3
/s in
September 2009) (USGS, 2011) and stormwater runoff, which are potentially large
source of heavy metals to the SCB (Schiff et al., 2000; Tiefenthaler et al., 2008).
In addition to point sources, desorption from suspended particles also appears to
be an important process influencing the concentration of some dissolved metals in the
coastal ocean off Los Angeles. This is evidenced by significant correlations between the
dissolved and the labile particulate pool for Cu and Pb (Figure A-3 in Appendix A). Pb
had the highest overall association between dissolved and labile pools (r
2
=0.61, all
stations) with stations having high particulate Pb values showing a stronger association
(r
2
= 0.92, stations with particulates ≥4.6 nM Pb) (Figure A-3B in Appendix A). Cu also
79

showed an association with 17 out of 29 stations occurring within a 95% confidence
interval of a linear regression (r
2
= 0.37; Figure A-3A in Appendix A). These associations
indicate that surface desorption from suspended particulates may be a source of dissolved
Cu and Pb. This trend was not seen for other trace metals including Cd, which had
virtually no association between labile and dissolved pools despite nearly 100% of Cd
particulates being labile in nature (Table A-2 in Appendix A). The sources of these
particulate trace metals in the SCB have primarily been associated with antifouling paint
for Cu and stormwater runoff and remobilization from sediments contaminated with Pb
before the elimination of unleaded gasoline. Deposition of Cu and Pb into the SCB has
resulted in enrichment of these metals in approximately 20% of the SCB area with metals
loads being especially high in some coastal regions such as the Palos Verdes Shelf,
harbors, and industrialized port areas (Phillips, 2007).

Temporal Gradients in Dissolved Trace Metals in the SCB Surface Waters
A comparison of dissolved metal concentrations measured in this study with those
measured in 1989 in the SCB (Sañudo-Wilhelmy and Flegal, 1992, 1994, 1996) suggests
that median metals levels in surface waters of the Bight have declined in general by a
factor of 2 for Ni, Cu, and Cd and by a factor of 3 for Ag, Co, and Pb (Figure 3-5).
Specifically, compared to the concentrations of trace metals measured in the SCB 20
years ago, average values are slightly lower for Cd, Cu, Ni, Ag, and Pb in 2009 (158 ± 15
pM Cd in 1989 vs 134 ± 36 nM in 2009; 2.4 ± 0.4 nM Cu in 1989 vs 1.3 ± 0.7 in 2009;
16 ± pM Ag in 1989 vs 5 ± 3 pM in 2009).
80

SCB 1989 SCB 2009 Punta Banda
Ag (pM)
0
5
10
15
20
SCB 1989 SCB 2009 Punta Banda
Pb (pM)
0
20
40
60
80
100
120
140
SCB 1989 SCB 2009 Punta Banda
Cd (pM)
0
200
400
600
800
1000
Upwelling
season
San Gabriel
river area
San
Gabriel
river area
Hyperion
area
San
Gabriel
river area
Santa Monica Bay
SCB 1989 SCB 2009 Punta Banda
Cu (nM)
0
1
2
3
4
5
6

Figure 3-5: Box-plots of dissolved Cu, Ag, Pb, and Cu concentrations measured in the SCB in 1989 and in
2009 and in Punta Banda, Mexico. SCB 1989 concentrations were measured at near shore stations from
Point Loma, Coronado, Imperial Beach, and the US-Mexico Border (Sañudo-Wilhelmy and Flegal, 1991,
1992, 1994, 1996). SCB 2009 values are all the metal concentrations measured in February and September
2009 in our area of study. Punta Banda concentrations include all the measurements in samples collected
every 2 weeks from March 2004−April 2005 ( Appendix A). The arrows identify specific locations or
oceanographic processes where or when the metal concentrations were significantly higher.

The range of dissolved metal concentrations measured in 2009 off Los Angeles
(1.2 pM − 1 3 pM Ag; 0.6 nM − 5 nM Cu; 93 pM − 252 pM Cd; 1.2 nM − 4.3 nM Ni)
were comparable and not significantly different than the concentrations measure in the
unpopulated area in Punta Banda, Mexico, used as a “control” uncontaminated region
(Figure 3-4). In fact, dissolved Cd and Fe were slightly higher on average in Punta Banda
(Cd 134 ± 36 nM in Los Angeles and 489 ± 118 nM in Punta Banda; Fe 1.2 ± 2.3 nM in
Los Angeles and 4.1 ± 2.1 nM in Punta Banda). The higher levels of these metals in the
Punta Banda area are most likely the result of strong upwelling that occurs on the shelf of
the Baja California Peninsula (Sañudo-Wilhelmy and Flegal, 1991; Zaytsev et al., 2003).
81

The Case of Lead
Our analysis of particulate and dissolved Pb concentrations in the coastal ocean
off Los Angeles suggests that surface water contamination of this toxic metal has been
reduced since the elimination of leaded gasoline and shows continuing decreases relative
to previous measurements performed in the late 1980s. Dissolved Pb concentrations were
last measured in this region in 1989 when Sa udo -Wilhelmy and Flegal (1994) reported a
3-fold decrease in Pb concentrations compared to the 1970s (Patterson et al., 1976;
Patterson, 1974). February 2009 samples were within a similar range of near shore SCB
samples collected in 1989 (mean 77 pM ± 45 in 1989 compared to mean 43 pM ± 18 in
February 2009). In contrast, September 2009 samples were markedly lower with 19/26
stations having dissolved Pb concentrations below our detection limit of 8 pM. Pb
concentrations had the largest difference between the two sampling months of all
measured trace metals, potentially due to the strong association of this trace element with
surface runoff, oceanic advection, and particle scavenging (Patterson, 1974; Sañudo-
Wilhelmy and Flegal, 1994). While the dissolved levels of Pb have been significantly
lowered, the legacy of Pb enriched particles from previous aeolian deposits and
wastewater discharge can result in periodic pulses of dissolved Pb from desorption from
suspended particulates (Figure 3-2, Figure A-3 in Appendix A) introduced to the water
column by stormwater runoff and sediments resuspension. The elevated Pb levels
measured near the San Gabriel River outfall in the February 2009 samples support the
aforementioned mechanism.

82

Rapid Internalization of Trace Metals in Synechococcus sp. CC9311
Trace metal additions to axenic cultures of Synechococcus sp. CC9311 resulted in
an increase in internal metal concentrations on average after 3 h of exposure for Cd
(+16%), Co (+55%), Fe (+26%), Ni (+15%), Mo (+38%), Cu (+29%), and Pb (+45%)
suggesting that many toxic metals can be introduced into the food chain within one tidal
cycle (Figure 3-6).
0hr 3hr 6hr 12hr
Cu:P (Total)
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
Cu:P (Internal)
0.00000
0.00002
0.00004
0.00006
0.00008
0.00010
0.00012
0.00014
0hr 3hr 6hr 12hr
Pb:P (Total)
0
1e-5
2e-5
3e-5
4e-5
5e-5
Pb:P (Internal)
0.0
2.0e-6
4.0e-6
6.0e-6
8.0e-6
1.0e-5
1.2e-5
1.4e-5
1.6e-5

Figure 3-6: Total (gray bars) and intracellular (black bars) Cu and Pb concentrations measured in
Synechococcus sp. CC9311. Trace metal values are normalized to P to account for variations in biomass.
Error bars are standard error of culture replicates.

83

Internalization continued through the 6-h time point for Cd (+28%), Ag (+31%), Co
(+60%), Fe (+58%), Ni (+59%), Mo (+49%), Cu (+58%), and Pb (+62%) with
internalization of Pb and Co continuing for 12 h (+77% for Pb and +78% for Co). Al and
Zn were not internalized during the course of the experiment. The exposure to the metal
spike resulted in mortality of Synechococcus sp. after 48 h of exposure. While the culture
media may have had a lower concentration of dissolved organic carbon (DOC) that could
influence metal toxicity due to organic complexation, we do not expect this to bias our
results as the majority of the metals used in our study are not strongly chelated by DOC
(e.g., Ag, Cd, Ni, Pb). Furthermore, we used low nanomolar additions during our
experiments, already lower than the micromolar concentrations usually used in this type
of bioassay. The rapid internalization of these metals in Synechococcus sp. indicates that
even small inputs of these metals into the marine environment can result in biological
uptake that has the potential for subsequent transfer up in the food web and thus
highlights the importance in controlling toxic metal contamination.

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88

CHAPTER FOUR: CONCLUSIONS AND FUTURE DIRECTIONS
In this dissertation, I examined the ambient distributions of B-vitamins and some
bioactive trace metals in contrasting marine environments and established the biological
impacts of these compounds on marine organisms and biological processes. This work
includes the measurement of dissolved B-vitamins in the Eastern Tropical South Pacific
(ETSP) in order to examine the chemical, physical, and biological controls of B-vitamin
distribution, an analysis of the B-vitamin thiamin on nitrogen fixation in unicellular
diazotrophs. Those studies were complemented with the measurement of dissolved and
particulate trace metals in an urban coastal environment in order to establish the current
state of metal contamination in the coastal ocean of Los Angeles metropolitan area.
While these studies have added to our understanding of the distribution of B-vitamins and
trace metals in different marine environment, they have also opened many new avenues
of research (discussed below).
In the ETSP we found that dissolved B-vitamins had a dynamic distribution both
geographically and with depth. In contrast to a recent study that linked B-vitamin
distributions to water masses (Sañudo-Wilhelmy et al., 2012), we did not find strong
relationships between dissolved B-vitamin distributions to water masses or microbial
abundance/basic diversity as expected due to their role in vitamin synthesis (Bertrand et
al., 2011, Croft et al., 2005, Croft et al., 2006). Based on these findings, I hypothesize
that taxonomic diversity and biological activity (such as bacterial respiration rates,
photosynthetic activity rates, and gene transcription rates) may be more important to the
distribution of dissolved B-vitamins than the abundance of microorganisms in the
oligotrophic waters of the ETSP. I suggest that future studies analyze ambient B-vitamin
89

distribution in respect to biological activity and also include field and culture uptake rates
of B-vitamins in order to more fully understand the biological controls of those organic
cofactor distributions in some regions of the world ocean. Our data also suggest that
dissolved B-vitamin distribution may be limited by the distribution of inorganic nutrients
(such as N & P) in some regions of the world ocean which may be addressed in the future
with deck board nutrient addition experiments.
Specific B-vitamin relationships I observed include the link between the amino
acid methionine and vitamin B
12
as well as the relationship between vitamin B
12
and
dissolved cobalt. As methionine is required by important marine organisms such as
Pelagibacter ubique and UCYN-A cyanobacteria (Tripp et al., 2010; Tripp et al., 2008),
understanding what controls the environmental distribution of this amino acid is of great
interest. Our data showed a strong correlation between the distribution of dissolved
vitamin B
12
and methionine in the ETSP, which I suggest could be due to the activity of
vitamin B
12
dependent methionine synthase. Future research should examine this
connection potentially through the environmental comparison of dissolved vitamin B
12

and the abundance of the B-
12
dependent methionine synthase protein in the marine
environment. In order to establish the environmental controls of vitamin B
12
distribution,
I compared the distribution of dissolved cobalt to dissolved B
12
as cobalt has been shown
to limit B
12
synthesis in other low cobalt regions (Panzeca et al., 2008). My results
showed some of the lowest reported values of cobalt in the marine environment as well as
overall low values of vitamin B
12
. However, an observable relationship between B
12
and
cobalt was only seen in a relatively nutrient rich coastal station, suggesting that dissolved
B
12
production may also be co-limited by nutrients (such as N and P), which were also
90

very low in this region. I suggest that future studies on the relationship between B
12
and
cobalt consider co-limitation scenarios.
In addition to these observed trends in the ETSP, I reported a strong correlation
between vitamin B
6
and chlorophyll in the upper transect of this region. I hypothesize
that B
6
may be produced in excess by photosynthetic organisms due to the requirement of
vitamin B
6
for chlorophyll synthesis and oxidative stress responses (Havaux et al., 2009;
Tsang et al., 2003). As this is the first reported relationship of B
6
and chlorophyll
reported in the field, I suggest that these findings be used as a direction for more in depth
research. For example, I suggest uptake assays of vitamin B
6
in the field and in culture
studies to determine if dissolved B
6
is being utilized by phytoplankton and/or bacteria in
the field. I also suggest that we need to elucidate the role of vitamin B
6
in photosynthesis
and nitrogen fixation due to the role of B
6
in oxidative stress responses.
Lastly in the ETSP, we observed an increase in chlorophyll and haptophytes with
a pigment signature matching Phaeocystis in field B
7
addition incubation in the upper
transect. This is the first ever-reported instance of vitamin B
7
increasing photosynthetic
biomass in the marine environment. In addition, Phaeocystis strains have not yet been
identified to be B
7
auxotrophs, and it has even been suggested that organisms with
complex plastids, such as haptophytes, do not require B
7
as a group (Croft et al., 2006). I
suggest that these findings may indicate that some haptopyhtes may in fact be B
7

auxotrophs and that further research should be conducted to establish if this is the case.
While the ETSP study was focused on understanding the controls of dissolved B-
vitamin concentrations, the study performed in Hawaii was focused on identifying the
role of B-vitamins on a specific biological process. Specifically, I identified two strains
91

of unicellular diazotrophs to be thiamin (vitamin B
1
) auxotrophs and showed that thiamin
increases nitrogen fixation rates in the field as well as in laboratory cultures of some
cyanobacteria. It was previously thought that inorganic nutrients such as phosphorous,
iron, and carbon dioxide (Mills et al., 2004; Monteiro et al., 2011; Sañudo-Wilhelmy et
al., 2001; Fu et al., 2008; Hutchins et al., 2007) were the controllers of nitrogen fixation
rates in the ocean. This work shows that organic compounds may also be involved in
controlling nitrogen fixation rates in the marine environment and adds to our
understanding of nitrogen fixation in the world ocean. I suggest that future research
further explore the role of thiamin on nitrogen fixation in other areas of the world ocean
and further suggest that the varying metabolic requirements for thiamin by unicellular
diazotrophs may also impact the community composition of these organisms in the field.
In addition to the impact of thiamin on nitrogen fixation rates, I showed that
diurnal profiles of dissolved thiamin have an overall inverse relationship between night
and day and that the concentrations vary with depth. Specifically, at mid-depth in the
euphotic zone I showed low levels of dissolved thiamin during the day and high levels of
dissolved thiamin at night. I suggest that this variability may impact the timing, vertical
distribution, and rate of nitrogen fixation by thiamin auxotrophs. Future research is
needed to further understand the nature of diurnal variability of dissolved vitamins in the
marine environment and to determine the impact this has on biological activity and
species composition.
While the first two studies in this dissertation focus on the nutrient properties of
dissolved B-vitamins and cobalt, I also examined the levels of dissolved and particulate
trace metals in an urban ocean environment in order to establish current levels of metal
92

contamination in this region. This study demonstrates an overall reduction in particulate
and dissolved trace metal concentrations in the coastal ocean off Los Angeles since the
implementation of the Clean Water Act and elimination of leaded gasoline in the United
States. I demonstrated that point sources such as waste water treatment plants seem to be
significant sources of trace metals to the surface waters off Los Angeles. Additionally, I
have established that the San Gabriel river system is a previously unidentified metal
source to the coastal ocean. Current metal contamination appears to be primarily
associated with the particulate phase and desorption from suspended particles seems to be
a potential source for Pb and Cu, both of which have elevated levels in local sediments
from decades of use in gasoline (Pb) and antifouling paint (Cu). In contrast to the
particulate metal pool, dissolved values of trace metals measured off Los Angeles are
now within the range of concentrations measured in the relatively pristine, unpopulated
area of Punta Banda, Mexico. Laboratory experiments showed that Synechococcus sp.
rapidly internalizes dissolved metals thereby suggesting that, despite their lowered
concentrations in the SCB, toxic metals might still get incorporated within the local food
web. We therefore suggest that future studies research the upward mobility of trace
metals in trophic levels and focus on the particulate phase of toxic metals in urban
environments.

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106

APPENDIX A: SUPPORTING INFORMATION FOR CHAPTER THREE
Table A-1: Approximate coordinates of 1976 near effluent and near shore stations.
Station Category Longitude Latitude
361 1976 near effluent (figure 1) -118.56 33.93
443 1976 near effluent(figure 1) -118.28 33.65
284 1976 near shore (figure S1) -118.84 34
304 1976 near shore (figure S1) -118.78 33.97
352 1976 near shore (figure S1) -118.6 33.81
444 1976 near shore (figure S1) -118.27 33.58
448 1976 near shore (figure S1) -118.09 33.52
525 1976 near shore (figure S1) -117.73 33.45
563 1976 near shore (figure S1) -117.67 33.41

Cd Zn Pb Ba Ni Cu V
Cd (ppm dry weight)
0
1
2
3
4
5
Zn, Pb, Ba, Ni, Cu (ppm dry weight)
0
5
10
15
20
25
30
35
V (ppm dry weight)
0
20
40
60
80

Figure A-1: Particulate metal concentrations for near shore stations in February 1976 (black) and February
2009 (grey). 1976 values are mean values of surface waters from stations 284, 304, 352, 444, 448, 525,
and 563 (Bruland & Franks, 1978) and 2009 stations are mean ± standard deviation of surface waters
samples from stations 2506, 2706, 3306, 4004, and 4001. 2009 stations were selected based on proximity to
1976 stations.

107
Table A-2. Total and % labile particulate trace metal concentrations of SCB study site surface waters in February 2009. Station numbers
correspond to environmental monitoring stations by the Los Angeles County sanitation district and the Los Angeles City sanitation department.
Station Longitude Latitude V % Fe % Co % Mo % Ni % Cu %
[nM] labile [nM] labile [pM] labile [nM] labile [nM] labile [nM] labile
2501 -118.12 33.73 23.0 20 8452 6.8 1907 5.5 0.9 4.8 6.1 5.5 7.7 26
2503 -118.14 33.67 13.0 11 4569 2.2 1211 2.7 0.98 6.0 3.4 10 5.7 30
2506 -118.16 33.58 8.0 2.9 84 5.2 121 20 11 9.2 1.8 29 2.3 44
2602 -118.19 33.69 26.0 18 8217 2.9 2019 2.5 4.9 15 6.2 8.9 9.5 32
2604 -118.2 33.64 9.7 0.59 206 1.1 90 15 13 18 1.8 22 1.1 51
2606 -118.22 33.59 1.5 0.41 59 3.5 39 45 4.3 15 0.4 64 1.1 82
2706 -118.27 33.61 7.6 2.2 93 2.9 66 16 7.9 15 1.7 21 1.3 57
2802 -118.29 33.69 14.0 13 2690 1.3 613 1.9 0.93 9.9 15 0.57 2.3 20
2805 -118.3 33.65 5.1 2.9 831 1.2 219 1.6 1.8 7.8 4.6 4.1 1.1 34
2903 -118.34 33.7 13.0 12 3141 1.9 776 4.3 1 6.4 17 1.2 2.4 21
2906 -118.35 33.65 3.8 2.3 484 2.6 123 6.3 1.9 12 2.7 5.7 0.67 51
3003 -118.37 33.71 25.0 23 8301 1.1 1760 6.4 1.5 3.2 45 1.0 6.7 12
3006 -118.39 33.67 6.3 2.4 378 1.3 163 10 4.7 9.2 2.6 15 0.76 71
3056 -118.43 33.69 15.0 14 4356 0 1076 2.7 2.8 3.2 23 1.1 3 4.2
3104 -118.45 33.75 1.2 0.86 56 0 72 39 0.44 5.8 0.73 23 1.8 6.5
3106 -118.48 33.71 4.9 1.4 98 0.85 79 27 2.6 21 1.1 18 0.27 54
3204 -118.44 33.84 7.6 2.2 182 1.9 10 0 5.2 35 0.65 29 0.64 43
3206 -118.49 33.81 5.4 2.0 139 1.2 79 15 3.6 12 1.1 18 0.8 40
3304 -118.46 33.88 4.6 1.6 266 2.2 73 0 1.8 33 0.4 38 0.34 100
3306 -118.53 33.85 2.4 0.92 137 1.8 89 24 2 9.7 1.4 18 0.87 61
3504 -118.49 33.92 0.6 0.31 116 1.8 BD BD 0.11 0 0.85 36 0.36 100
3606 -118.56 33.92 0.6 0.34 131 1.8 BD BD BD BD 0.37 100 0.36 100
3704 -118.53 33.97 9.1 7.6 3257 3.6 608 0.8 1 2.2 4.1 27 5.9 53
3802 -118.59 34.03 2.1 2.1 92 78.5 91 71 0.21 16 0.66 56 0.99 93
3804 -118.6 33.99 0.4 0.15 52 2.2 4 0.0 0.21 0 0.98 48 0.35 100
108

3806 -118.62 33.96 0.4 0.23 79 0.84 37 12 0.16 13 0.9 21 0.26 68
3904 -118.72 34 0.6 0.32 111 2.6 52 0 0.23 0 1.3 36 1 47
3906 -118.72 33.94 2.2 0.73 93 1.5 68 33 0.95 14 1.1 30 2.6 15
4001 -118.81 34 11.0 10 2603 2.5 559 3.8 1.5 7.7 3.3 12 2.1 32
4004 -118.81 33.96 0.8 0.44 124 2.4 16 0 0.13 0 0.38 35 0.32 62

Table A-2: Continued
Station Longitude Latitude Cd % Pb % Zn % Ba % Al %
[pM] labile [pM] labile [nM] labile [nM] labile [nM] labile
2501 -118.12 33.73 83 32 10717 54 19 26 75 3.3 1191 1.3
2503 -118.14 33.67 55 84 3657 51 13 32 48 8.9 318 1.1
2506 -118.16 33.58 153 100 365 87 6.8 48 10 67 7.4 0
2602 -118.19 33.69 100 70 5234 51 19 31 114 12 669 1.2
2604 -118.2 33.64 110 100 291 77 4.8 55 22 74 9.6 0
2606 -118.22 33.59 61 100 248 100 2.7 72 4.8 77 2.6 0
2706 -118.27 33.61 65 100 212 74 2 66 4.9 82 2.8 0
2802 -118.29 33.69 92 23 1107 40 5.4 23 34 4.9 228 0.57
2805 -118.3 33.65 31 100 558 60 4.4 39 16 38 57 0.54
2903 -118.34 33.7 72 65 1425 47 8.2 23 42 7.9 186 0.81
2906 -118.35 33.65 49 100 337 75 4.5 54 13 44 26 1.1
109

3003 -118.37 33.71 137 75 4615 23 15 13 122 4.7 385 0.5
3006 -118.39 33.67 102 100 465 72 6.7 52 22 50 28 0.1
3056 -118.43 33.69 161 100 1255 7 8.3 11 51 1.2 122 0
3104 -118.45 33.75 90 100 72 100 2.8 26 1.1 39 1.8 0
3106 -118.48 33.71 116 100 104 97 0.9 34 9.1 76 5.2 0
3204 -118.44 33.84 99 100 262 100 1.5 86 76 44 10 0
3206 -118.49 33.81 43 100 219 79 2.7 36 20 71 8.7 0
3304 -118.46 33.88 28 100 374 88 1.9 83 44 59 17 0.17
3306 -118.53 33.85 114 100 193 76 2.6 42 8.6 67 8.3 0
3504 -118.49 33.92 93 100 272 100 0.9 100 2.6 58 2.7 0
3606 -118.56 33.92 72 100 343 100 0.9 100 1.5 25 2.3 0
3704 -118.53 33.97 163 100 5483 68 16 66 37 20 71 1.4
3802 -118.59 34.03 66 97 1610 100 6.1 77 3.2 92 6.4 61
3804 -118.6 33.99 195 100 193 100 1.4 85 0.81 46 1.7 0
3806 -118.62 33.96 73 100 69 100 2.5 32 1.6 23 4.4 0
3904 -118.72 34 349 100 354 100 1.5 100 1.8 36 2.8 0
3906 -118.72 33.94 147 100 115 100 4.8 21 11 59 3.5 0
4001 -118.81 34 198 78 1751 40 6.1 40 25 15 146 1.1
4004 -118.81 33.96 130 100 167 100 1.4 100 2.4 47 5.9 0
110
Table A-3. Procedural blanks and detection limits for particulate trace metal analysis. Blanks are the
average of filter blanks with the highest and lowest values not included. Detection limits are 3x the
standard deviation of the blank values.

Detection
limit
(pM)
(Labile)
Procedural
Blank
(pM)
(Labile)
Detection
limit
(pM)
(Refractory)
Procedural
Blank
(pM)
(Refractory)
V
0.08 0.1 0.72 0.85
Fe
8.53 17 317 343
Co
0.47 0.60 0.25 0.42
Mo
0.68 0.45 0.5 2.7
Ni
1.49 2.5 2.4 22.3
Cu
2.7 4.1 7.2 9.2
Cd
0.07 0.05 0.27 0.32
Pb
0.07 0.25 0.32 0.90
Zn
5.6 7.0 12 11
Ba
1.5 1.6 2.7 2.6
Al
10 27 833 567

111
Table A-4: Dissolved trace metal concentrations of SCB study site surface waters in February 2009. Station numbers correspond to environmental
monitoring stations by the Los Angeles County sanitation district and the Los Angeles City sanitation department.

Station Longitude Latitude V Fe Co Mo Ni Cu Cd Ag Pb Zn Al
[nM] [nM] [pM] [nM] [nM] [nM] [pM] [pM] [pM] [nM] [nM]
2501 -118.12 33.73 14 7.5 945 86 4.3 5 208 13 100 8.6 BD
2503 -118.14 33.67 16 2.8 202 89 2.5 3.2 146 12 62 2.8 63
2506 -118.16 33.58 35 0.86 128 86 2.4 1.4 142 7.2 66 1.2 BD
2604 -118.2 33.64 37 0.65 98 80 2 1 114 4.4 39 1 52
2606 -118.22 33.59 39 1.1 123 82 2.1 1.4 135 6.2 62 1.4 71
2706 -118.27 33.61 21 0.72 135 90 2.6 1.5 125 7.9 46 1 42
2802 -118.29 33.69 34 2.4 140 80 2.2 1.9 143 8.9 53 2.6 BD
2805 -118.3 33.65 39 1.3 109 84 2.1 1.3 131 7.8 44 1.1 BD
2903 -118.34 33.7 46 2.5 142 96 2.2 1.7 147 10 39 1.7 119
2906 -118.35 33.65 37 1.7 143 81 2.4 1.6 156 11 51 2.1 BD
3003 -118.37 33.71 33 3.1 173 81 2.3 1.5 172 9 38 1.3 BD
3006 -118.39 33.67 33 0.91 62 78 2 1.1 110 6.2 39 0.95 BD
3056 -118.43 33.69 39 0.58 34 85 2.1 0.71 73 2.1 31 0.21 94
3104 -118.45 33.75 20 0.84 47 92 2.4 0.74 81 4.5 31 0.26 BD
3106 -118.48 33.71 1.1 BD 30 98 3.1 0.84 86 11 31 0.13 101
3204 -118.44 33.84 35 0.72 72 81 1.7 1 118 7.5 37 0.42 39
3206 -118.49 33.81 23 0.63 75 94 2.5 1.4 98 5.2 35 0.49 48
3304 -118.46 33.88 43 1.2 93 90 2.1 1.3 123 12 62 1.1 BD
3306 -118.53 33.85 25 1 77 99 2.9 1.5 109 12 37 0.61 98
3504 -118.49 33.92 35 1.2 57 86 2 1 95 4.7 29 0.29 85
3505 -118.53 33.91 31 0.59 50 76 2 0.9 115 3 27 0.29 BD
3606 -118.56 33.92 32 0.51 41 79 1.8 0.61 87 2.3 25 0.42 42
3704 -118.53 33.97 34 2.4 121 80 2 1.7 137 9.8 79 2.3 82
3804 -118.6 33.99 31 0.48 39 77 1.9 0.68 93 2.2 29 0.19 44
3806 -118.62 33.96 33 0.35 40 79 1.9 0.79 78 2 27 0.29 75
112

3904 -118.72 34 37 0.91 44 80 1.8 0.65 93 2.6 25 0.25 96
3906 -118.72 33.94 42 0.9 49 89 2.3 1 97 2.6 33 0.25 39
4001 -118.81 34 36 2.6 120 94 2.3 1.1 135 6.3 36 0.32 BD
4004 -118.81 33.96 35 1.2 68 79 2.1 0.78 128 3.1 26 0.22 47

113

Table A-5: Dissolved trace metal concentrations of SCB study site surface waters in September 2009. Station numbers correspond to environmental
monitoring stations by the Los Angeles County sanitation district and the Los Angeles City sanitation department.

Station Longitude Latitude V Fe Co Mo Ni Cu Cd Ag Pb Al
[nM] [nM] [pM] [nM] [nM] [nM] [pM] [pM] [pM] [pM]
2503 -118.14 33.67 15 0.46 57 58 1.8 1.3 54 2.4 7.6 BD
2506 -118.16 33.58 24 0.34 49 74 2.2 1.1 75 2.4 8.7 BD
2602 -118.19 33.69 17 0.53 54 55 1.7 1.2 67 2.1 BD BD
2604 -118.2 33.64 14 0.22 40 45 1.2 0.79 34 1.7 BD BD
2606 -118.22 33.59 19 0.57 52 61 1.8 0.78 71 3 BD 142
2802 -118.29 33.69 17 1.2 165 55 2.5 3 86 4.1 BD BD
2805 -118.3 33.65 18 0.25 50 58 1.9 0.85 68 2 BD BD
2903 -118.34 33.7 16 0.67 78 64 1.8 1.6 65 3.1 BD BD
2906 -118.35 33.65 20 0.61 54 63 2 1.4 66 2.7 7.6 BD
3003 -118.37 33.71 21 0.37 74 62 1.9 1.1 68 2 BD BD
3006 -118.39 33.67 24 0.2 55 73 1.7 1.3 71 3.8 BD BD
3056 -118.43 33.69 17 BD 53 52 1.7 0.9 75 3.3 BD BD
3104 -118.45 33.75 24 0.39 86 73 2.2 1.9 93 6.4 14 BD
3106 -118.48 33.71 22 0.22 37 78 1.9 0.9 65 2.2 BD 189
3204 -118.44 33.84 49 1.5 172 169 4.1 2.4 147 6.9 12 198
3206 -118.49 33.81 22 0.25 46 66 1.9 0.89 70 2.6 BD BD
3505 -118.53 33.91 21 0.29 29 64 2 2 59 2.8 BD BD
3704 -118.53 33.97 17 0.22 50 65 2.1 0.84 61 1.3 BD BD
3802 -118.59 34.03 16 1.7 75 62 2.1 1.1 96 3.4 BD BD
3804 -118.6 33.99 22 BD 41 66 2 0.77 63 1.2 BD BD
3806 -118.62 33.96 13 3.5 55 52 1.7 0.73 85 1.9 BD BD
3904 -118.72 34 18 0.75 66 64 2 0.95 86 2 BD BD
3906 -118.72 33.94 14 12 96 55 2 1.1 252 2.5 29 BD
4001 -118.81 34 22 0.63 81 69 2 1.1 81 3.1 BD BD
114

4004 -118.81 33.96 17 0.34 56 64 1.9 0.89 70 1.7 BD BD
4006 -118.81 33.91 21 0.65 57 68 2.1 0.92 78 3.8 BD BD
115
Table A-6. Procedural blanks and detection limits for dissolved trace metal analysis. Blanks are the
average of Milli-Q® blanks with the highest and lowest values not included. Detection limits are 3x the
standard deviation of the blank values.

Detection limit
[pM]
Febraury 2009
Procedural Blank
[pM]
February 2009
Detection limit
[pM]
September 2009
Procedural Blank
[pM]
September 2009
V
12 18 22 53
Fe
157 247 292 180
Co
5.4 3.3 3.9 4.0
Mo
77 120 176 525
Ni
115 79 19 9.0
Cu
31 32 22 39
Cd
5.9 8.1 3.4 2.0
Pb
0.89 0.39 7.6 4.7
Zn
35 26 - -
Ag 0.64 0.34 0.58 1.01
Al
59 33 153 142

116

Table A-7. Dissolved trace metal concentrations of bi-weekly samplings in Punta Banda, Mexico (33º N,
117º W) from 2004-2005.

Date V
[nM]
Fe
[nM]
Co
[pM]
Ni
[nM]
Cu
[nM]
Cd
[pM]
Ag
[pM]
Pb
[pM]
3/16/2004 31 6.5 72 2.9 0.49 2.6 80
3/31/2004 26 3.5 51 3.2 1.2 0.55 3.2 56
4/16/2004 25 2.1 44 2.1 1.1 0.41 1.8 26
4/30/2004 31 4.2 51 2.7 1.4 0.41 3.4 26
5/14/2004 21 2.0 22 1.6 0.65 0.29 2.1 15
5/31/2004 28 3.1 37 3.3 0.84 0.49 2.4 15
6/15/2004 28 2.7 28 3.0 1.1 0.46 1.9 36
6/30/2004 28 6.3 145 2.4 1.0 0.53 2.1 20
7/15/2004 30 7.8 92 2.9 1.1 0.54 1.7 9
7/30/2004 27 4.3 41 2.0 0.83 0.44 2.5 11
8/16/2004 35 7.6 77 3.0 1.3 0.57 3.3 18
9/1/2004 27 5.7 48 2.2 1.1 0.43 7.1 10
9/15/2004 36 8.3 72 3.3 1.4 0.60 8.2 39
9/30/2004 32 66 2.6 1.0 0.49 3.7 35
10/13/2004 34 7.5 109 4.5 2.2 0.60 10 57
11/1/2004 36 5.9 77 3.8 1.5 0.52 8.1 40
11/15/2004 25 3.0 56 2.1 0.67 0.37 3.9 16
11/30/2004 29 2.8 69 2.5 0.77 0.46 4.6 23
12/14/2004 23 2.3 67 2.6 0.82 0.39 5.2 18
1/5/2005 28 3.4 70 2.4 0.86 0.42 5.0 17
1/17/2005 29 2.3 102 3.3 2.1 0.46 6.1 53
2/1/2005 33 1.9 81 3.0 1.1 0.49 4.2 23
2/15/2005 27 1.9 90 2.6 1.2 0.40 5.1 16
3/7/2005 30 3.3 129 3.8 1.4 0.53 6.7 20
3/31/2005 31 2.2 58 3.1 0.84 0.47 4.5 22
4/15/2005 41 3.0 114 11 1.6 0.85 12 21
4/29/2005 36 136 4.5 1.5 0.77 12 25

117

Iron[nM]: February 2009 (>0.45 m)
Vanadium [nM]: February (>0.45 m)
Cobalt[pM]: February 2009 (>0.45 m)
Nickle [nM]: February (>0.45 m)

Figure A-2: February 2009 surface water particulates for Co, Ni, Fe, V, Mo, and Zn.
118

Molybdenum[nM]: February 2009 (>0.45 m)
Zinc [nM]: February (>0.45 m)
Aluminum [nM]: February 2009 (>0.45 m)

Figure A-2: Continued

119

Pb labile [pM]
0 1000 2000 3000 4000 5000 6000 7000
Pb dissolved [pM]
20
40
60
80
100
120
140
B
Labile Cu (nM)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Dissolved Cu (nM)
0
1
2
3
4
5
6
A

Figure A-3: Plot of dissolved and labile particulate Cu from February 2009 samples and dissolved and
labile particulate Pb from February 2009 samples. Error lines represent 95% confidence interval.

120

Iron [nM]: February 2009 (<0.45 m)
Iron [nM]: September 2009 (<0.45 m)
Cobalt [pM]: February 2009 (<0.45 m) Cobalt [pM]: September 2009 (<0.45 m)

Figure A-4: Dissolved trace metal maps of Fe, Ni, V, Mo, and Co for February and September 2009
stations
121

Vanadium [nM]: February 2009 (<0.45 m)
Vanadium [nM]: September 2009 (<0.45 m)
Nickle [nM]: February 2009 (<0.45 m)
Nickle [nM]: September 2009 (<0.45 m)

Figure A-4: Continued

122

SCB 1989 SCB 2009 Punta Banda
Ni (nM)
0
2
4
6
8
10
12
SCB 1989 SCB 2009
V (nM)
10
20
30
40
50
60
SCB 1989 SCB 2009 Punta Banda
Co (pM)
0
200
400
600
800
1000
SCB 1989 SCB 2009
Fe (nM)
0
2
4
6
8
10
12
14

Figure A-5: Dissolved Ni, V, Co and Fe in SCB stations and Punta Banda, Mexico. SCB 1989 Ni and Co
are near shore values from Point Loma, Corando, and Imperial Beach (Sañudo-Wilhelmy & Flegal, 1996).
SCB 2009 values include February and September samples and Punta Banda values include samples
collected every 2 weeks from March 2004 – April 2005.

123

0hr 3hr 6hr 12hr
Al:P (Total)
0.0
0.2
0.4
0.6
0.8
1.0
Al:P (Internal)
0.00
0.02
0.04
0.06
0.08
0.10
0hr 3hr 6hr 12hr
Ni:P (Total)
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
Ni:P (Internal)
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
0.0012
0.0014
0hr 3hr 6hr 12hr
Zn:P (Total)
0.0
0.1
0.2
0.3
0.4
Zn:P (Internal)
0.000
0.002
0.004
0.006
0.008
0.010
0hr 3hr 6hr 12hr
Co:P (Total)
0.0
2.0e-6
4.0e-6
6.0e-6
8.0e-6
1.0e-5
1.2e-5
1.4e-5
1.6e-5
1.8e-5
Co:P (Internal)
0.0
5.0e-6
1.0e-5
1.5e-5
2.0e-5
2.5e-5
0hr 3hr 6hr 12hr
Fe:P (Total)
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
Fe:P (Internal)
0.000
0.002
0.004
0.006
0.008
0.010
0hr 3hr 6hr 12hr
Cd:P (Total)
0
1e-5
2e-5
3e-5
4e-5
Cd:P (Internal)
0.0
2.0e-6
4.0e-6
6.0e-6
8.0e-6
1.0e-5
1.2e-5
1.4e-5
1.6e-5
1.8e-5

Figure A-6: Total (grey) and internal (black) Ni, Co, Fe, Cd, Al, and Zn in Synechococcus sp. CC9311.
Trace metal values are normalized to phosphorous to account for variations in biomass. Error bars are
standard error of culture replicates.

Abstract (if available)

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Asset Metadata

Creator Smail, Emily Ann (author)

Core Title B-vitamins and trace metals in the Pacific Ocean: ambient distribution and biological impacts

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Marine and Environmental Biology

Publication Date 11/07/2012

Defense Date 10/12/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag auxotrophy,B-vitamin,Clean Water Act,nitrogen fixation,OAI-PMH Harvest,thiamin,trace metal,unicellular diazotroph,urban ocean,water contamination

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Sañudo-Wilhelmy, Sergio A. (committee chair), Berelson, William M. (committee member), Caron, David A. (committee member), Hutchins, David (committee member), Webb, Eric A. (committee member)

Creator Email easmail1@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-107651

Unique identifier UC11289092

Identifier usctheses-c3-107651 (legacy record id)

Legacy Identifier etd-SmailEmily-1273.pdf

Dmrecord 107651

Document Type Dissertation

Rights Smail, Emily Ann

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