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The impact of the concentration and distribution of dissolved and particulate B-vitamins and their congeners on marine microbial ecology
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The impact of the concentration and distribution of dissolved and particulate B-vitamins and their congeners on marine microbial ecology
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Content
© Copyright 2017 by Christopher P. Suffridge
All Rights Reserved
The impact of the concentration and distribution of
dissolved and particulate B-vitamins and their congeners
on marine microbial ecology
Christopher P. Suffridge
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 2017
© Copyright 2017 by Christopher P. Suffridge
All Rights Reserved
Approved by
Advisory Committee
Sergio A. Sañudo-Wilhelmy (Chair)
David A. Caron
William M. Berelson
iii
Table of Contents
Table of Contents ....................................................................................................... iii
Chapter 1 Introduction ............................................................................................ 1
Background ..................................................................................................... 1 1.1
1.1.1 B-Vitamin Structure and Function ............................................................ 2
1.1.2 B-Vitamin Auxotrophy in Marine Systems ............................................... 5
1.1.3 The Oceanography and Ecology of B-Vitamins ........................................ 7
Contents of this Dissertation ......................................................................... 9 1.2
References ..................................................................................................... 12 1.3
Chapter 2 A New Analytical Method for Direct Measurement of Particulate
and Dissolved B-Vitamins and their Congeners in Seawater ............................ 16
Abstract ......................................................................................................... 17 2.1
Introduction .................................................................................................. 17 2.2
Methods ......................................................................................................... 19 2.3
2.3.1 Culture Sample Collection, Processing, and Preservation ...................... 19
2.3.2 Environmental Sample Collection, Processing, and Preservation ........... 20
2.3.3 Particulate B-Vitamin Extraction, and Purification ................................. 21
2.3.4 Dissolved Sample Preconcentration ........................................................ 22
2.3.5 LC/MS Analysis ...................................................................................... 23
Results ........................................................................................................... 25 2.4
2.4.1 Particulate Method Validation ................................................................. 25
2.4.2 Dissolved Method Validation .................................................................. 27
iv
2.4.3 Internal Standard ...................................................................................... 28
Discussion ...................................................................................................... 29 2.5
References ..................................................................................................... 39 2.6
Chapter 3 The potential role of dissolved and particulate B-vitamins and their
congeners influencing microbial diversity in the Mediterranean Sea. ............. 42
Abstract ......................................................................................................... 43 3.1
Introduction .................................................................................................. 44 3.2
Methods ......................................................................................................... 47 3.3
3.3.1 Cruise Track ............................................................................................ 47
3.3.2 B-vitamin Sample Collection .................................................................. 48
3.3.3 B-vitamin Analysis .................................................................................. 48
3.3.4 Biomass Parameters Sample Collection and Analysis ............................ 49
3.3.5 N* Calculation ......................................................................................... 49
3.3.6 Statistical Analysis .................................................................................. 50
Results and Discussion ................................................................................. 50 3.4
3.4.1 Dissolved B-Vitamin Concentrations ...................................................... 54
3.4.2 Dissolved B-Vitamin Congener Relative Abundance ............................. 58
3.4.3 Particulate and Dissolved B-Vitamin Pool Partitioning .......................... 63
3.4.4 Particulate B-Vitamin Congener Relative Abundance ............................ 66
3.4.5 B-Vitamin Quotas .................................................................................... 70
3.4.6 Dissolved B-Vitamin Based Linear Regression Modeling ...................... 78
Conclusions ................................................................................................... 87 3.5
Acknowledgements ....................................................................................... 89 3.6
v
References ..................................................................................................... 90 3.7
Chapter 4 B-vitamin intracellular quotas influence marine microbial bloom
succession. .............................................................................................................. 98
Abstract ......................................................................................................... 99 4.1
Introduction ................................................................................................ 100 4.2
Methods ....................................................................................................... 104 4.3
4.3.1 Sample collection .................................................................................. 104
4.3.2 Incubation setup ..................................................................................... 104
4.3.3 Experimental sampling .......................................................................... 105
4.3.4 B-vitamin sample collection .................................................................. 105
4.3.5 B-vitamin analysis ................................................................................. 106
4.3.6 Heterotrophic bacteria and picoautotroph quantification ...................... 107
4.3.7 Eukaryotic plankton diversity ................................................................ 107
4.3.8 Specific growth rates ............................................................................. 107
4.3.9 Chlorophyll A (Chl-a) quantification .................................................... 108
4.3.10 Elemental (C, N, P) quantification ...................................................... 108
4.3.11 Statistical analysis ................................................................................ 109
Results and Discussion ............................................................................... 110 4.4
4.4.1 Background conditions at SPOT ........................................................... 110
4.4.2 B-Vitamin Co-Limitation ...................................................................... 118
4.4.3 B-vitamin Mediated Bloom Succession ................................................ 124
4.4.4 Particulate B-Vitamin Concentrations: Uptake v. Synthesis ................. 128
4.4.5 Estimated intracellular B-vitamin concentrations ................................. 131
vi
4.4.6 B-Vitamin Quotas within Specific Experimental Treatments ............... 134
4.4.7 B-vitamin Synergy: B
12
quotas in all treatments ................................... 144
Conclusion ................................................................................................... 147 4.5
Acknowledgements ..................................................................................... 149 4.6
References ................................................................................................... 150 4.7
Chapter 5 Conclusions and Future Directions .................................................. 157
1
Chapter 1 Introduction
Background 1.1
Phytoplankton are major components of the global environment and are
responsible for about half of the global primary production (Field et al. 1998).They
support the base of the oceanic food web, and mediate carbon flux from the atmosphere
to the deep ocean via the biological carbon pump (Chisholm 2000). Phytoplankton are of
such importance to the global environment that much research has been conducted to
elucidate the factors that control their growth and community structure. For example,
during the past several decades, biogeochemical studies have focused primarily on the
role that inorganic macronutrients and some trace metals play on regulating
phytoplankton dynamics in the world ocean (Boyd et al. 2000; Bruland and Lohan 2003;
Dugdale and Goering 1967; Falkowski et al. 1998; Kolber et al. 1994; Martin et al. 1994;
Mills et al. 2004). However, the pioneering work of several investigators carried out
between the 1950s through the 1970s showed that macronutrients and trace metals are not
the exclusive factors influencing the microbial community, and that the availability of B-
vitamins has the potential to regulate the microbial ecology and thus biogeochemical
cycles in the ocean (Carlucci 1970; Carlucci and Silberna 1966c; Droop 1957b; Menzel
and Spaeth 1962; Provasoli 1958; Ryther and Guillard 1962; Sanudo-Wilhelmy et al.
2014). The relatively small body of knowledge existing on the distribution of dissolved
B-vitamins in the ocean, combined with physiological studies of the effects of B-vitamins
on phytoplankton dynamics, suggests that these compounds are not present in sufficient
quantities to support maximal phytoplankton production (Bertrand et al. 2012; Bertrand
2
et al. 2011; Bertrand et al. 2007; Koch et al. 2011; Koch et al. 2013; Panzeca et al. 2008;
Panzeca et al. 2006; Sañudo-Wilhelmy et al. 2012; Sañudo-Wilhelmy et al. 2006).
Therefore, in order to fully understand the degree to which B-vitamins control marine
microbial ecological processes, we must be able to constrain the cycling of B-vitamins
between the dissolved and the particulate pools in the ocean. To our knowledge, no
current data on the distributions of particulate B-vitamins in the ocean exist.
Furthermore, all the marine studies on B-vitamins to date have been on cyanocobalamin,
thiamin, and biotin. The concentrations of multiple important biochemical congeners of
thiamin and cobalamin (Figure 1.1) have never been measured in either the particulate or
the dissolved pool in marine systems. Without measuring these important bioactive and
inactive congeners it is impossible to gain a full understanding of the total available pool
of B-vitamins. The research presented in this dissertation will provide, for the first time,
coupled particulate and dissolved oceanic distributions of B-vitamins and their
biochemical congeners, establish intracellular B-vitamin quotas for important functional
groups of organisms, and will allow us to further understand the role that B-vitamins and
their congeners play in influencing phytoplankton bloom succession in marine systems.
1.1.1 B-Vitamin Structure and Function
B-vitamins are essential coenzymes that play a role in metabolic pathways across
all domains of life. The functions of these compounds are highly conserved and it has
been hypothesized that some B-vitamins served as ancient pre-enzymatic metabolic
catalysts (Monteverde et al. 2017). The B-vitamins are defined operationally as water-
soluble coenzymes, so the structure and function of each B-vitamin varies greatly.
Vitamins B
1
(thiamin), B
7
(biotin), and B
12
(cobalamin) are the B-vitamins that are most
3
commonly required by phytoplankton and bacterioplankton (reviewed by Sañudo-
Wilhelmy et al. 2014). Multiple biochemical congeners of these vitamins exist, including
both bioactive and bioinactive forms (Figure 1.1). Organisms have the ability to readily
remodel and salvage these congeners to obtain the active form of the B-vitamin (Begley
1996; Begley et al. 1999; Chatterjee et al. 2006; Edwards et al. 2017; Gray and Escalante-
Semerena 2009; Kraft and Angert 2017; Yi et al. 2012). Therefore it is essential to
measure multiple B-vitamin congeners, both in the dissolved and particulate pools, in
order to gain a full understanding of the availability of these coenzymes and the
biochemical processes they mediate.
Figure 1.1 Molecular structures of B-vitamin congeners. Congeners in boldface text are bioactive forms.
Thiamin (Vitamin B
1
) is found in three chemical forms: unphosphorylated thiamin (B
1
),
thiamin monophosphate (TMP), and thiamin pyrophosphate (TPP) (Figure 1.1). The
Cobalamin-B
12
R-Groups
R
Adenosyl-B
12
Methyl-B
12
R-CH
3
Cyano-B
12
R-CN
Hydroxy-B
12
R-OH
Thiamin-B
1
Figure 2. Thiamin Synthesis From Thiazole and Pyrimidine Precursors
Thiamin synthesis by bacteria, plants, and fungi requires independent production or acquisition of a thiazole
precursor hydroxyethylthiazole (HET) phosphate and a pyrimidine precursor 4-amino-2-methyl-5-hydroxy-
methylpyrimidine (HMP) pyrophosphate that combine to form thiamin monophosphate, which is phosphory-
lated to become the active form of the molecule that serves as the cofactor thiamin pyrophosphate (TDP). See
the online edition for a color version of thisfigure.
THIAMIN INFLUENCE ON ECOLOGICAL INTERACTIONS June 2017 155
This content downloaded from 068.181.207.012 on May 16, 2017 09:06:46 AM
All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c).
HMP
TMP TPP
Thiamin
Biotin-B
7
Methionine
Scheme1. Two Met synthases are known to catalyze this reaction: cobalamin-
dependent methionine synthase, the so-called metH gene product in Escherichia coli,
andcobalamin-independentmethioninesynthase,themetEgeneproduct.Homologues
of MetH are found in animals, including mammals, and in many species of eubacteria
andsimpleeukaryotes,butnotinplantsorArchaea.HomologuesofMetEarefoundin
plants,insects,yeast,Archaea,andmanyspeciesofeubacteria,butnotinmammals.In
the MetE family, Me transfer appears to result from direct attack of Hcy on CH
3
-
H
4
folate
6
); the enzymes do not contain organic prosthetic groups. Me Transfer
catalyzed by MetH involves the cobalamin prosthetic group, and transfer occurs from
methylcobalamintoHcy,leadingtomethionineandcob(I)alamin,andthenfromCH
3
-
H
4
folate to cob(I)alamin to regenerate the methylcobalamin cofactor and form
H
4
folate.
Tertiary amines are not good Me donors; the pK
a
of the corresponding H
4
folate
anionic leaving group is estimated to be !30. The available evidence suggests that
activation of CH
3
-H
4
folate occurs by protonation at N(5); in solution, the pK
a
of the
resulting ammonium compound is 5.05 [1]. Thus, at physiological pH, only ca. one
moleculein100isinthecorrectprotonationstateforreaction.ThemicroscopicpK
a
of
theSHgroupofHcyis10[2],andthethiolassuchisnotnucleophilic.Thus,onlyonein
1000molecules of Hcy will be in the reactive thiolate form at neutral pH.
Afurthercomplicationisthat,inaqueoussolution,protonatedCH
3
-H
4
folatewould
reactwiththeHcythiolatebyH
!
transferratherthanbygrouptransfer.Thisisavoided
in −improbable×MetHbytheinterpositionofthecobalamincofactorintheMe-transfer
sequence, i.e.,CH
3
-H
4
folate and Hcy do not contact one another in the course of the
reaction. But in the −impossible× reaction catalyzed by MetE, H
!
transfer between
substratesmustbeavoided.Asimplesolutiontothiscatalyticdilemmawouldbeforthe
enzymestolowerthepK
a
ofHcybelow7,whileraisingthepK
a
ofCH
3
-H
4
folateabove
7,maximizing theconcentration ofthereactiveforms of thesubstratesandminimizing
Scheme1. Reaction Catalyzed by Methionine Synthases. The MetE enzyme appears to catalyze a direct attack
oftheS-atomofHcyontheN(5)MegroupofCH
3
-H
4
folate,whilemethyltransfercatalyzedbyMetHinvolves
the cobalamin cofactor as an intermediary. MetH can use substrates with one or more Glu residues (n"1),
while MetE requires at least three Glu residues (n"3).
!"#$"%&'()*&+&'(,'%( ± Vol.86 (2003) 3940
6
) MetE requires three or more Glu residues in its CH
3
-H
4
folate substrate (CH
3
-H
4
Pte(Glu)
n
) for efficient
catalysis, while MetH can use CH
3
-H
4
folate substrates with one or more Glu residues. The Glu residues in
CH
3
-H
4
Pte(Glu)
n
are connected by amide linkages involving the !-carboxylate of the preceding residue.
4
active form of thiamin is TPP, which acts via stabilizing the acyl carbanion in ketone
rearrangement reactions in central metabolism as shown in Figure 1.2. This vitamin is an
coenzyme for over thirty enzymes including transketolase (carbon fixation) and pyruvate
dehydrogenase (entry into the TCA cycle; Figure 1.2). Thiamin is synthesized through
the ligation of a thiazole and pyrimidine moieties (Begley et al. 1999; Chatterjee et al.
2006). It has been shown that the pyrimidine moiety (HMP) is required by the
ecologically important SAR11 clade of bacteria in order to fulfill its thiamin requirement
(Carini et al. 2014). Biotin (vitamin B
7
) catalyzes carboxylation and decarboxylation
reactions in central metabolism and fatty acid metabolism (Figure 1.2). It is a coenzyme
for about ten enzymes, and it is the only B-vitamin to be covalently bonded to its
enzymes (Waldrop et al. 2012). Cobalamin (vitamin B
12
) has four major biochemical
congeners including two bioactive (adenosylcobalamin (AB
12
) and methylcobalamin
(MB
12
)) and two bioinactive (hydroxycobalamin (HB
12
) and cyanocobalamin (CB
12
))
forms (Figure 1.1) (Brown 2005). Prokaryotes are able to readily interconvert these
corrinoids, and then regenerate them after catalysis (Gray and Escalante-Semerena 2009;
Koutmos et al. 2009; Yi et al. 2012), which makes measuring all four chemical forms of
heightened importance. Cobalamin is a coenzyme for two major classes of enzymes:
AB
12
mediated isomerases, where the Co-C bond cleaved homolytically producing a
radical, and MB
12
mediated methyltransferases where the Co-C bond is heterolytically
cleaved (Banerjee and Ragsdale 2003). Vitamin B
12
plays a vital role in the synthesis of
the amino acid methionine, which is the first amino acid in essentially all peptides
(Figure 1.2) (Dowling et al. 2012; Matthews et al. 2003).
5
Figure 1.2 Central metabolic pathways with vitamin-requiring steps highlighted. Red arrows are B
1
-
dependent steps, purple arrows are B
7
-dependent steps, and blue arrows are B
12
-dependent steps. Specific
enzyme names can be found by referring to the EC numbers in the EXPASY enzyme database. Figure is
adapted from Sañudo-Wilhelmy et al. 2014.
1.1.2 B-Vitamin Auxotrophy in Marine Systems
Pioneering research on B-vitamins in marine systems was carried out four
decades ago (Carlucci and Bowes 1972; Carlucci and Silberna 1966c; Droop 1957a;
Droop 1957b; Menzel and Spaeth 1962; Provasoli 1958; Ryther and Guillard 1962).
These researchers used bioassay methods to determine the concentrations of vitamins B
1
,
B
7
, and B
12
dissolved in seawater and within some eukaryotic phytoplankton cultures
under laboratory conditions. Much of this initial work was done in conjunction with
MA06CH13-SanudoWilhelmy ARI 12November2013 14:4
Glucose-6-P
Pyruvate
Glycolysis or
gluconeogenesis
Acetyl-CoA
α–Ketoglutarate
Ribonucleotides
RNA
DNA
Deoxyribonucleotides
Ribulose-P
Glyceraldehyde-3-P
Calvin-Benson
cycle and/or
pentose phosphate
pathway
3-Phosphoglycerate
Aspartate
family AA
(Met)
Ser and
Cys
EC 1.17.4.2
Ala, Leu,
and Val
Phosphoenolpyruvate
Fructose-6-P
Ribose-P
Fatty acid synthesis
Trp, Tyr,
and Phe
EC 6.4.1.2
EC 2.2.1.1
Succinyl-CoA Methylmalonyl-CoA
Oxalacetate
EC 2.1.1.13
EC 5.4.99.2
EC 1.2.4.2
EC 2.2.1.6
EC 1.2.4.1
EC 4.1.1.3
EC 6.4.1.1
EC 4.1.2.22
Krebs
cycle
Porphyrins
Figure 4
Centralmetabolicpathwayswithvitamin-requiringstepshighlighted.RedarrowsareB
1
-dependentsteps,
purplearrowsareB
7
-dependentsteps,andbluearrowsareB
12
-dependentsteps.Specificenzymenamescan
befoundbyreferringtotheECnumbersinTable 3.
what is known about the ecology of Bacteroidetes, seem to imply that, as a group, Bacteroidetes
ismoremetabolicallysimilarthanProteobacteria.
There are 12 B
7
-dependent enzymes, 8 of which were found in the surveyed marine taxa
(Figure 3b, Table 3). All of these enzymes are predicted to be B
7
-dependent carboxylases
(Knowles1989).ThemostcommonandwidespreadB
7
-dependentenzymefoundwasacetyl-CoA
carboxylase(Figure3b).Thisenzymewaspresentinatleast85%ofourtaxonomicgroupsandin
100%oftheCyanobacteria,Alphaproteobacteria,andBacteroidetesgroups.Interestingly,itwas
also the only B
7
-dependent enzyme found in Cyanobacteria. In addition to its recently defined
role in archaeal CO
2
fixation, acetyl-CoA carboxylase is essential for fatty acid synthesis in most
described microbes; thus, it is crucial for energy storage as well as the production of important
cellularcomponents,suchasmembranes(Jitrapakdee&Wallace2003).
www.annualreviews.org • The Role of B Vitamins in Marine Biogeochemistry 353
Annu. Rev. Marine. Sci. 2014.6:339-367. Downloaded from www.annualreviews.org
by Christopher Suffridge on 01/08/14. For personal use only.
FIGURE I.1
Figure adapted from Sañudo-Wilhelmy et al. 2014 Ann. Rev. Marine Science
6
early attempts to bring phytoplankton into laboratory culture using synthetic growth
media. Research was focused on vitamins B
1
, B
7
, and B
12
because these B-vitamins have
the highest incidences of auxotrophy (obligate requirement) among eukaryotic
phytoplankton. These early culture based studies led to the conclusion that B-vitamin
auxotrophy is widespread amongst the eukaryotic phytoplankton (Croft et al. 2006;
Provasoli and Carlucci 1974; Tang et al. 2010). Out of more than 300 species of
eukaryotic phytoplankton studied both by culture and genome sequencing approaches,
more than half require B
12
, almost a quarter require B
1
, and only about 8% require B
7
(Figure 1.3) (Sanudo-Wilhelmy et al. 2014).
Marine bacteria were not known to be important to global biogeochemical cycles
at the time of the early work with B-vitamins, so they were not included in the original
studies carried out between the 1950s and 1970s. Recent genome mining projects have
discovered that 76%, 78%, and 37% of marine bacteria have the complete synthetic
pathways for vitamins B
1
, B
7
, and B
12
respectively (Figure 1.3) (Sanudo-Wilhelmy et al.
2014). These new results imply that like the eukaryotic phytoplankton, many prokaryotes
require exogenous sources of B-vitamins. Available genomes of dominant marine
microbial taxonomical groups were queried for B-vitamin requiring enzymes to
determine if the organisms have a requirement for the same B-vitamins that they cannot
independently synthesize (Gomez-Consarnau et al. 2017). The data showed that in all
cases, each taxonomic group (including those dominated by auxotrophs) contained at
least one enzyme requiring vitamin B
1
, B
7
, or B
12
, which indicates that B-vitamin
auxotrophy and metabolic requirements are widespread in marine systems (Sanudo-
Wilhelmy et al., 2014).
7
Figure 1.3 Percentages of marine species that synthesize (based on whole-genome sequencing) or require
(based on culture experiments) B vitamins. The sequencing analyses are described in more detail in Sañudo-
Wilhelmy et al. 2014. The numbers of B-vitamin-requiring species are from culture data compiled by Tang et al.
(2010). Abbreviations: HMP, 4-amino-5-hydroxymethylpyrimidine; THZ, 4-methyl-(β-hydroxyethyl) thiazole.
Figure is adapted from Sañudo-Wilhelmy et al. 2014.
1.1.3 The Oceanography and Ecology of B-Vitamins
A few ocean transects reporting spatial gradients in dissolved B-vitamin
concentrations have been conducted (Sañudo-Wilhelmy et al. 2012; Smail 2012). In all
cases, the concentrations of the dissolved B-vitamins are extremely low, within the
picomolar range. In addition, there appear to be large swaths of ocean where the
concentrations of dissolved B-vitamins are below limits of detection in the femptomolar
range (Sañudo-Wilhelmy et al. 2012). In fact, several studies published recently suggest
that the small concentrations of available dissolved B-vitamins may be insufficient to
support maximal production in the systems studied (Bertrand et al. 2012; Bertrand et al.
2011; Bertrand et al. 2007; Gobler et al. 2007; Koch et al. 2012; Koch et al. 2011; Koch
et al. 2013; Panzeca et al. 2008; Panzeca et al. 2006; Sañudo-Wilhelmy et al. 2006).
These studies demonstrate the importance of B-vitamins, such as B
1
and B
12
, on the
FIGURE I.2
Figure adapted from Sañudo-Wilhelmy et al. 2014 Ann. Rev. Marine Science
MA06CH13-SanudoWilhelmy ARI 12November2013 14:4
B
1
B
7
B
12
THZ HMP
0
10
20
30
40
50
60
70
80
90
100
Organisms surveyed (%)
0
152
180
324
10
25
327
5
88
314
5
88
Sequenced bacteria that synthesize B vitamins (n = 413)
Sequenced phytoplankton that synthesize B vitamins (n = 10)
Cultured phytoplankton that require B vitamins (n = 332)
Figure 2
Percentagesofmarinespeciesthatsynthesize(basedonwhole-genomesequencing)orrequire(basedon
cultureexperiments)Bvitamins.Thesequencinganalysesaredescribedinmoredetailin Tables 1and 2.
ThenumbersofB-vitamin-requiringspeciesarefromculturedatacompiledbyTangetal.(2010).
Abbreviations:HMP,4-amino-5-hydroxymethylpyrimidine;THZ,4-methyl-(β-hydroxyethyl)thiazole.
do not. Furthermore, the findings that some algae excrete substantial quantities of vitamins add
thealgaetotherosterofvitaminsourcesintheocean.
Toevaluatevitaminauxotrophyinprokaryotes,wedeterminedthepresenceorabsenceofthe
vitamin synthesis pathways of more than 400 species of marine bacteria, with a special focus on
the microbial taxa most commonly found in marine environments: Cyanobacteria, Alpha- and
Gammaproteobacteria, and Bacteroidetes. Because these whole-genome-sequenced representa-
tives are cultured, their physiologies may differ from those of in situ uncultured communities.
Nevertheless,theyaregoodmodelorganismstopredicttheecologicalfunctionsofabundantma-
rinemicrobialtaxa(e.g.,whethertheyarevitaminsynthesizersorconsumers).Overall,76%,78%,
and37%ofthemarinebacteriahaddenovopathwaysforsynthesizingB
1
,B
7
,andB
12
,respectively
(Figure2).Thesedataimplythatmanymarinebacteriarequirevitaminsforgrowthandtherefore
mustcompetewithothermarineorganismsforthosevitamins.Asthedivisionratesofbacteriacan
befasterthanthoseofphytoplankton,bacteriamaybethemostimportantconsumersofBvitamins
intheocean,asKochetal.(2012)recentlydemonstratedempiricallyinsomemarineenvironments.
ThemembersofAlphaproteobacteriamakeupanextremelydiverseclass,rangingfromobligate
oligotrophicbacteriaintheSAR11clustertothosethatgrowonhighorganicmatterconcentra-
tionslike Rhodobacterales.BacteriaintheSAR11clusterarethemostabundanttaxonintheocean,
accounting for more than 60% of the sequences in the Global Ocean Sampling project (Rusch
et al. 2007). All sequenced SAR11 genomes lack de novo pathways for synthesizing vitamins B
1
,
B
7
andB
12
(Table2).Itisimportanttopointout,though,thattheentirevitaminB
1
biosynthetic
www.annualreviews.org • The Role of B Vitamins in Marine Biogeochemistry 345
Annu. Rev. Marine. Sci. 2014.6:339-367. Downloaded from www.annualreviews.org
by Christopher Suffridge on 01/08/14. For personal use only.
8
ecological function of different marine systems. The essential role of B-vitamins in both
primary and secondary metabolic pathways makes it clear that these compounds play a
vital role in affecting intracellular physiological processes.
Distributions of particulate/intracellular B-vitamins in the open and/or coastal ocean
have never before been measured simultaneously with the dissolved B-vitamin pool.
These measurements are required to understand how auxotrophic microbes can survive in
marine areas where dissolved B-vitamins are depleted. Additionally, neither the
dissolved nor the particulate concentrations of the multiple B-vitamin congeners
discussed above have ever been measured in the ocean. In order to gain a greater
understanding of the role these compounds play in controlling the phytoplankton
community structure and activity, we must first take steps to understand how they are
distributed in the environment.
The actual B-vitamin requirements (as cellular quotas) of both eukaryotic
phytoplankton and marine bacteria are not known. It has been shown that these
organisms require B-vitamins through both culture based and genome based studies,
however, to our knowledge, no work has been conducted to directly measure cellular
quotas for B-vitamins in the marine system. Carlucci and Bowes (1972) reported
intracellular concentrations of vitamins B
1
, B
7
, and B
12
from some eukaryotic
phytoplankton cultures, but their bioassay method may not have been able to adequately
detect these compounds (Carlucci and Bowes 1972; Carlucci and Silberna 1966a;
Carlucci and Silberna 1966b; Carlucci and Silberna 1966c). The directly measured
intracellular B-vitamin quotas that do exist are from bacterial model systems
(Escherichia coli) (Winkler et al. 2002). However, due to the diversity of metabolic
9
schemes present in the marine environment (e.g., photoautotrophy, chemoautotrophy,
heterotrophy, etc.), these loosely established quotas are not necessarily applicable to
marine systems. In order to further establish the cycling of B-vitamins it is imperative
that a technique is developed to directly measure the intracellular concentrations of B-
vitamins. The use of a technique that measures the particulate/intracellular B-vitamin
pool will allow us to complement the existing dissolved B-vitamin technique, and will
allow, for the first time ever, simultaneous measurements of dissolved and particulate B-
vitamins in any aquatic environment. These measurements are an important first step for
understanding the B-vitamin requirements of microbes in the ocean. Future research
using this method will allow for a greater understanding of the degree to which B-
vitamins control the dynamics of phytoplankton growth and community structure, which
will allow for a greater understanding of the global biological carbon pump among other
biologically-mediated processes.
Contents of this Dissertation 1.2
This dissertation is divided into three main chapters where the influence of B-
vitamins on marine microbial ecology is discussed. Chapter 2, entitled A New Analytical
Method for Direct Measurement of Particulate and Dissolved B-vitamins and Their
Congeners in Seawater, describes a new LCMS-based method to determine the
concentrations of B-vitamins and their congeners in the dissolved and particulate pool in
the ocean. This method forms the foundation of the entire dissertation, as prior to its
development, no technique existed to determine the concentrations of B-vitamins and
their congeners in these two fractions. Chapter 3, entitled The potential role of dissolved
and particulate B-vitamins and their congeners influencing microbial diversity in the
10
Mediterranean Sea, applies the new analytical method described in Chapter 2 in an
oceanographic setting that ranged from oligotrophic to eutrophic conditions, providing
the first ever paired dissolved and particulate B-vitamin measurements in any marine
system. Also, for the first time, the chemical congeners of the B-vitamins were measured
in these pools, providing a more complete understanding of the total B-vitamin
availability, and their impact on the ecology of a major marine basin. B-vitamin quotas
were also determined for all congeners that provide a novel insight to the physiological
demands of the microbial community for these growth factors. Additionally, linear
regression modeling was used to correlate the abundances of organisms with known B-
vitamin requirements or synthesis capability to the ambient dissolved B-vitamin
concentrations. These models provide a strong link between B-vitamin congener
availability and microbial community structure. Chapter 4, entitled B-vitamin
intracellular quotas influence marine microbial bloom succession, explores the impacts
of B-vitamin additions on species succession in a coastal upwelling system within the
Southern California Bight at the San Pedro Ocean Time-series (SPOT). We observed
that diatoms were co-limited by nutrients and cobalamin, while picoeukaryotes were co-
limited by nutrients and thiamin. B-vitamin quotas determined from these experiments
indicate that thiamin and biotin are predominantly synthesized while cobalamin is
predominantly assimilated from the dissolved pool. We observed strong differentiation
among the cobalamin congener relative abundance between the fractions containing
bacterioplankton (picoplankton fraction) and eukaryotic phytoplankton (nanoplankton
fraction). The active forms of cobalamin (AB
12
and MB
12
) had greater relative
abundances in the nanoplankton fraction, which might suggest that the lack of the de
11
novo synthesis pathway in eukaryotes has created different inter-domain cobalamin
requirements. Based on our findings, we hypothesize that B-vitamins control the species
succession in coastal upwelling systems. Finally, in the last chapter of this dissertation,
the major findings are summarized, and the potential future directions for research in this
field are discussed.
12
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15
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16
Chapter 2 A New Analytical Method for Direct Measurement
of Particulate and Dissolved B-Vitamins and their Congeners
in Seawater
By Christopher P. Suffridge
Coauthors: Lynda Cutter and Sergio A. Sañudo-Wilhelmy
Published as
Suffridge C, Cutter L, Sañudo-Wilhelmy S. A New Analytical Method for Direct
Measurement of Particulate and Dissolved B-Vitamins and their Congeners in Seawater.
Frontiers in Marine Science. 2017;4(11).
17
Abstract 2.1
We have developed a new method to directly measure the concentration of the particulate
and dissolved pools of B-vitamins in seawater. B-vitamins are coenzymes required for life, yet
many organisms cannot synthesize these compounds de novo and must scavenge them from the
environment. It has been shown that B-vitamins can control the marine microbial community’s
structure and activity; however, the actual B-vitamin requirements/quotas for marine microbes
are not well studied due to the lack of analytical protocols. This method will enable the study of
B-vitamin cellular quotas as well as their environmental cycling. With this method, we can also
simultaneously determine the biochemical congeners of vitamins B
1
and B
12,
as well as vitamins
B
2
, B
6
, B
7
, and the amino acid methionine in both the particulate and dissolved pool, using liquid
chromatography-mass spectrometry after a chemical extraction (particulate) or resin
preconcentration (dissolved). Particulate and dissolved B-vitamin concentrations were
simultaneously determined from a microbial community in the Atlantic Ocean. Particulate B-
vitamin concentrations in the Atlantic ranged from 0.01 pM (cyano-B
12
) to 46.4 pM (thiamin
monophosphate, a B
1
congener) while dissolved B-vitamin concentrations ranged from 0.07 pM
(adenosyl-B
12
) to 679.4 pM (B
7
). The application of this technique in different marine systems
has the potential to shed new light on previously unknown biochemical processes occurring
within the oceanic microbial community, and the resulting roles B-vitamins play in regulating
global biogeochemical cycles.
Introduction 2.2
B-vitamins are chemically diverse, water soluble coenzymes that are essential for all
forms of life (Berg et al. 2007). In the ocean there is a complex relationship between organisms
that are capable of de novo B-vitamin synthesis and those that require these compounds, or their
18
precursors, but are unable to synthesize them (Paerl et al. 2015; Sañudo-Wilhelmy et al. 2014).
Despite the known B-vitamin requirements of many climate-relevant eukaryotic phytoplankton,
including species of diatoms, dinoflagellates, and coccolithophores (Croft et al. 2006; Sañudo-
Wilhelmy et al. 2014; Tang et al. 2010), dissolved concentrations of these compounds are
remarkably low, ranging from undetectable to hundreds of picomoles per liter of water (Heal et
al. 2014; Sañudo-Wilhelmy et al. 2012; Suárez-Suárez et al. 2011). It has been shown that these
concentrations are in some cases too low to support maximal microbial production in many
regions of the world ocean, and that addition of B-vitamins can cause dramatic shifts in the
microbial community structure and production (Bertrand and Allen 2012; Bertrand et al. 2012;
Bertrand et al. 2013; Gobler et al. 2007; Koch et al. 2011; Sañudo-Wilhelmy et al. 2006). While
the importance of B-vitamins to phytoplankton has been well known for decades (Carlucci and
Silberna 1969; Droop 1962; Pinto et al. 2002; Provasoli and Carlucci 1974), never before have
the particulate B-vitamins from the marine microbial community and dissolved B-vitamins from
the surrounding water been simultaneously and directly measured. The lack of a technique to
directly measure the particulate pool of B-vitamins coupled with insufficient existing methods to
measure multiple biochemical forms of some B-vitamins (congeners) in the dissolved pool, has
hindered our understanding of their role in shaping the community composition of phytoplankton
species that influence carbon cycling, as well as the processes controlling B-vitamin cycling
between the dissolved and particulate pools in the ocean.
We have developed a method to directly determine the concentrations of particulate B-
vitamins in marine systems. In addition, we describe the modification of an existing dissolved B-
vitamin method to include important biochemical congeners of the B-vitamins (Heal et al. 2014;
Sañudo-Wilhelmy et al. 2012; Suárez-Suárez et al. 2011). The utilization of our new analytical
19
protocol will allow for a better understanding of B-vitamin distributions in the ocean, and the
roles they play in mediating global primary productivity and microbial species succession. The
main focus of our method is to measure multiple biochemically relevant congeners of vitamins
B
1
and B
12
which exhibit significant levels of auxotrophy (obligate requirement) among
eukaryotic phytoplankton (Croft et al. 2006; Sañudo-Wilhelmy et al. 2014; Tang et al. 2010).
This method quantifies three forms of thiamin: unphosphorylated thiamin (B
1
hereafter), thiamin
monophosphate (TMP), thiamin pyrophosphate (TPP); a thiamin precursor, the pyrimidine
moiety (4-amino-2-methyl-5-pyrimidinyl methanol, HMP); biotin (B
7
); and four forms of
cobalamin (B
12
): adenosylcobalamin (AB
12
), cyanocobalamin (CB
12
), hydroxycobalamin (HB
12
),
and methylcobalamin (MB
12
). In addition to the primary target analytes listed above, this method
can be used to quantify riboflavin (B
2
), pyridoxal (B
6
), and the amino acid methionine (Met).
Taken together, these analytes represent different active forms and precursors of an important
subset of B-vitamins, which have essential biochemical roles in central and secondary metabolic
pathways across all domains of life (Begley et al. 1999; Brown 2005; Waldrop et al. 2012). This
method simultaneously measures the particulate and dissolved pools of chemically and
functionally diverse B-vitamins that are commonly required by marine microbes, thus enabling
novel insights into the ways that B-vitamins impact marine microbial community structure and
production.
Methods 2.3
2.3.1 Culture Sample Collection, Processing, and Preservation
Cultures of Synechococcus strain CC9311 and Vibrio strain AND4 were used to optimize
the particulate extraction protocol. CC9311 was grown in vitamin free SN media under 24hr
illumination (100µE) at 20°C (Waterbury et al. 1986). AND4 was grown in Zobell media at
20
room temperature (Gomez-Consarnau et al. 2010). Cultures were grown under axenic conditions
however we did not formally test for axenicity at the end of the experiment since these cultures
were only used for a method development and not for a physiological study. Cells were collected
in late exponential/early stationary growth phase via gentle vacuum filtration onto 25mm
diameter, 0.2µm pore size Supor filters (Pall Biosciences). 10ml of cell culture was filtered.
Cellular density was assessed using direct enumeration with epifluorescence microscopy (Hobbie
et al. 1977; Waterbury et al. 1986). Filters were placed in 2ml cryovials (Nalgene) and
immediately frozen and stored at -80°C until particulate vitamin quantification.
2.3.2 Environmental Sample Collection, Processing, and Preservation
Environmental samples used for verification of both the particulate and dissolved
methods were collected from two locations: the San Pedro Ocean Time series (SPOT) station
(33°33’N, 118°24’W, Figure 2.1) off the coast of Southern California in March 2015, and a
station in the Eastern Atlantic Ocean (33°1’16”N, 12°14’28”W, Figure 2.1) in May of 2014.
Samples were collected from 3m at SPOT and from four depths in the Atlantic: 3m, 50m, 85m
(deep chlorophyll maximum), and 200m. All samples were filtered using a peristaltic filtration
system. The peristaltic filtration rig was cleaned between samples with methanol to remove any
residual B-vitamins. Prior to use, the rig was rinsed thoroughly with Milli-Q water followed by
sample seawater (Sañudo-Wilhelmy et al. 2012). Particulate samples were screened through a
10µm mesh and filtered onto 0.2µm Supor filters. After filtration was completed, the filters were
frozen and stored as described for the cultured samples. Particulate organic carbon (POC)
samples were collected in parallel with environmental particulate B-vitamin samples in order to
allow normalization of the particulate B-vitamin concentrations. They were filtered on
precombusted GF/F filters under low vacuum pressure, desiccated at 60°C, and analyzed using a
21
CHN elemental analyzer (Knap et al. 1996). The filtrate resulting from the particulate fraction
filtration was collected for the dissolved B-vitamin analysis in 1L amber HDPE bottles and
stored at -20°C until analysis (Okbamichael and Sañudo-Wilhelmy 2004; Okbamichael and
Sañudo-Wilhelmy 2005; Sañudo-Wilhelmy et al. 2012).
Figure 2.1 Map of environmental sampling locations. A) The San Pedro Ocean Timeseries (SPOT) is located within
the Southern California Bight (33°33’N, 118°24’W). B) The Atlantic Station was located north of the Canary Islands and
west of Morocco (33°1’16”N, 12°14’28”W).
2.3.3 Particulate B-Vitamin Extraction, and Purification
Frozen filters with samples collected from both cultures and the environmental samples
were aseptically transferred from the cryovials into acid-washed, methanol-rinsed, 15ml heavy-
walled polypropylene conical centrifuge tubes, containing 4ml of cold lysis solution with 2ml of
0.5mm zirconia/silica beads (Biospec). The lysis solution consisted of 5% methanol, 95%
LC/MS-grade water solution adjusted to pH 3.5 using hydrochloric acid. The ratio of extraction
solution to beads was 2:1 v/v solution:beads. The exact volume of the lysis solution was
determined using an analytical balance. Cells were lysed via bead beating by vortexing on high
for 5 minutes followed by 1 minute in an ice bath to ensure that the temperature of the solution
did not exceed 30°C. This process was repeated 6 times, for a total of 30 min of bead beating to
achieve complete cellular lysis, which was determined by epifluorescence microscopy.
Following cell lysis, the solution was incubated for 30 minutes in the dark at 30°C to extract the
22
target analytes from the cellular matrix. Organic-aqueous liquid phase extractions (LPE) were
used to remove hydrophobic cellular components from the cellular lysate, while simultaneously
purifying the hydrophilic target analytes. Chloroform (1:1 v/v) was added to each tube, and the
tubes were vigorously shaken for 3 minutes causing the hydrophobic cellular components to
partition into the chloroform. The tubes were then centrifuged at 5000 rpm for 10 minutes to
achieve complete phase separation, and to pelletize large cellular debris (Mashego et al. 2007;
Roberts et al. 2012). The upper aqueous phase containing the B-vitamins was transferred to a
new tube, and the chloroform extraction process was repeated twice. Following the liquid phase
extraction, samples were adjusted to pH 6.5 by adding dilute NaOH. Sample volume was
assessed before and after addition of NaOH using an analytical balance in order to correct
concentrations for the added volume. Samples were centrifuged for 10 minutes at 5000rpm and
then syringe push filtered through methanol-rinsed 0.2µm Minisart RC4 matrix filters (Sartorius
Stedim Biotech).
2.3.4 Dissolved Sample Preconcentration
Samples for dissolved vitamin analysis were preconcentrated as has been previously
described (Okbamichael and Sañudo-Wilhelmy 2004; Okbamichael and Sañudo-Wilhelmy 2005;
Sañudo-Wilhelmy et al. 2012). Briefly, preconcentration columns were prepared by pouring 1:1
MeOH: C
18
resin (HF Bondesil (Agilent Technologies)) slurry into Poly-Prep Columns (Biorad).
The resin was allowed to settle, and the excess MeOH was drained leaving 7ml of resin. Thawed
samples were adjusted to pH 6.5 using dilute HCl and passed over the preconcentration column
at 1ml per min. Samples were then adjusted to pH 2.0 using dilute HCl and were passed over the
preconcentration column a second time. The residual salt was rinsed off the resin using 30ml of
LC/MS grade water. The target analytes were then eluted from the resin using 12ml of LC/MS
23
grade methanol into methanol-rinsed 15ml conical centrifuge tubes. Eluted samples were then
further concentrated via evaporation in a nitrogen dryer using 5-20PSI compressed N
2
gas, in the
dark, at room temperature. Samples were allowed to evaporate until 300µl remained. The
samples were then stored at -20°C until LC/MS analysis which occurred within 24hours. Prior to
LC/MS analysis samples were adjusted to pH 6.5 using 10µl of dilute NaOH. Care was taken to
measure the mass of the sample before and after addition of the base and used to correct the final
concentrations. Two different pH papers were used to assess pH after the final mass had been
recorded. This is possible because the initial pH of each sample is known (e.g., pH 2.0 after
preconcentration, pH 3.5 for the particulate samples) so the amount of base to be added can be
calculated. Samples were centrifuged and push filtered as described above. Previous tests
confirm that no vitamins adsorb onto the Minisart filters used for push filtering.
2.3.5 LC/MS Analysis
Quantification of B
1
, TMP, TPP, HMP, B
2
, B
6
, B
7
, AB
12
, CB
12
, HB
12
, MB
12
, and Met
was carried out with a Thermo TSQ Quantum Access electrospray ionization triple quadrupole
mass spectrometer, coupled to a Thermo Accela High Speed Liquid Chromatography (LC/MS)
system. The LC system used a stable-bond C
18
reversed-phase column (Discovery HS C
18
10cm
x 2.1mm, 5µm column, Supelco Analytical) with a 100µL sample loop. In order to increase the
sensitivity and precision of our analysis, the LC/MS was run in full-loop mode (100µl/injection).
The computer software Excalibur and LCQuan (Thermo Scientific) were used for data
acquisition and analysis. A 12-minute gradient flow was used with mobile phases of methanol
(MeOH) and LC/MS grade water, both buffered to pH 4 with 0.5% acetic acid. The flow rate
was set at 230 µl/min throughout the run, with a gradient starting at 93% LC/MS water: 7%
MeOH for two minutes, changing to 100% MeOH by seven minutes, and continuing at 100%
24
MeOH until nine minutes and returning to initial conditions until the gradient completes at
twelve minutes. All peaks were identified using standards dissolved in LC/MS grade water;
retention times are listed in Table 2.1. The mass spectrometer was run in Selected Reaction
Monitoring (SRM) mode with positive polarity with a well time of 100ms per transition. The
resolution of the mass filters used for quadrupoles 1 and 2 were 0.7 and 0.1m/z respectively. The
ESI spray voltage was 4000V, sheath gas (N
2
) pressure was 30PSI, auxiliary gas (Ar) pressure
was 3PSI, capillary temperature was 269°C, and the collision pressure was 2.1 torr. The mass to
charge ratio (m/z) of the analytes and their respective product ions, as well as the analyte specific
collision energy and T-lens settings are listed in Table 2.1. The instrumental limits of detection
(LOD; Table 2.2) were calculated as three times the standard deviation of the procedural blank
(if no signal was detectable in the analytical blank, then they were calculated as three times the
standard deviation of the lowest detectable standard). The resulting data was analyzed using the
software environment R, and all figures were created using the Ggplot2 software package for R
(R Core Team 2015; Wickham 2009).
Table 2.1 Analytical LC/MS Parameters for all measured analytes. Relative retention time is the time the peak
appears relative to the internal standard used for quantification (e.g., the TPP peak is 0.3 min after the B
1
internal
standard peak).
Analyte
SRM Parent-
Product m/z
Collision
Energies
(V) T-Lens
Column
Retention
Time (min)
Relative
Retention
Time (min)
IS
used RF
Thiamin 265 - 81,122,144 15,15,14 70 1.4 0 B1 1.00
Thiamin monophosphate 345 - 122,126,224 17,29,35 86 1.4 0 B1 0.49
Thiamin pyrophosphate 425 - 122,126,304 21,34,16 78 1.7 0.3 B1 3.94
Pyrimidine moiety 139 - 122,81,54 11,18,31 60 1.7 0.3 B1 2.04
Riboflavin 377 - 172,197,242 34,37,22 87 8.0 0 B2 1.00
Biotin 245 - 97,123,227 30,26,13 87 7.4 -0.6 B2 0.03
Adenosylcobalamin 791 - 147,665 38,20 98 7.7 -0.3 B2 0.61
Cyanocobalamin 678 - 147,359,997 40,26,40 91 7.4 -0.6 B2 0.02
Hydroxycobalamin 665 - 147,359,636 33,17,14 91 7.5 -0.5 B2 0.07
Methylcobalamin 673 - 147,359,665 47,17,19 92 7.9 -0.1 B2 0.31
Methionine 150 - 56,61,104 10 74 2.3 0.9 B1 0.96
Thiamin IS 269 - 81,122,144 10 42 1.4
Riboflavin IS 383 - 174,202,249 35,36,22 176 8.0
25
Results 2.4
2.4.1 Particulate Method Validation
The particulate B-vitamin extraction method was initially optimized using cultures of
Synechococcus str. CC9311 and exclusively focusing on vitamins B
1
, B
7
, and CB
12
. These
analytes were chosen due to the widespread auxotrophy for these compounds in marine
microbial systems (Sañudo-Wilhelmy et al. 2014). As a starting point we used the vitamin
extraction protocol described by Carlucci and Bowes (1972) (100°C incubation for 1hour in
seawater adjusted to pH 3.5), however this protocol resulted in vitamin degradation as our spike
recoveries were below 10%. In order to increase those recoveries, extractions were conducted
with iteratively milder (both temperature and pH) conditions until it was found that bead beating
in a lysis solution composed of 5% LC/MS grade methanol and 95% LC/MS grade water
adjusted to pH 3.5 did not degrade any of our analytes, while also still achieving complete
cellular lysis (determined by epiflourescence microscopy). This resulted in greater than 90%
spike recoveries for our three targeted analytes (Table 2.2). Initially, a second extraction of the
filter was conducted to ensure complete extraction, and no signal was observed on this
subsequent extraction. Based on the high spike recoveries obtained for the targeted analytes, our
analytical scope was then successfully expanded to include all analytes listed in Table 2.2. In
addition to those analytes, we attempted to analyze a chemical form of thiazole (C
3
H
3
NS), a B
1
precursor, however our spike recoveries were only 60%, and a signal was only occasionally
observed within the particulate pool. It is unclear if our method could be used to measure
pseudocobalamins (Helliwell et al. 2016; Tanioka et al. 2009) and other vitamin precursors such
as the B
1
precursor 4-methyl-5-thiazoleethanol (Paerl et al. 2015). We focused on analytes that
had standards readily commercially available at the time of method development, and limited our
26
analytical scope to preserve analytical sensitivity and precision. Future studies, potentially
focusing solely on one class of analytes (e.g. cobalamins and pseudocobalamins) will be needed
to evaluate these, and other, B-vitamin congeners and precursors with sufficient sensitivity and
precision. However, the high recoveries of the added analytes shown in Table 2.2 indicate that
this extraction method can likely be expanded to a wide range of chemically diverse metabolites.
Table 2.2 Analyte spike recoveries and instrumental limits of detection (L.O.D). The L.O.D. was calculated as three
times the standard deviation of the blank, and when no signal was present in the blank, three times the standard deviation
of the lowest detectable concentration of standards was used. Procedural limits of detection depend on the
preconcentration volume, extraction volume, or the volume of sample filtered, which vary between each sample. They
range from 3-6 orders of magnitude lower than the instrumental L.O.D.
When Synechococcus biomass was initially extracted and analyzed, it became apparent
that sample matrix suppression was occurring as evidenced by low recoveries, poor peak shape,
and a substantial increase in LC/MS background noise. Both liquid phase extractions (LPE) and
solid phase extractions (SPE) were tested to reduce the analytical signal to noise ratio by
purifying our analytes of interest from the total cellular extract. The SPE used C
18
resin, and
resulted in full recoveries of our analytes, but did not satisfactorily reduce the background noise.
The LPE using chloroform (1:1 v/v) did not degrade our analytes, and no vitamins were observed
Analyte
% Particulate
Spike Recovery
% Dissolved
Spike Recovery L.O.D. (nM)
Thiamin 98.6 ± 10.4 99.9 ± 16.2 5.6
Thiamin monophosphate 99.1 ± 7.6 89.7 ± 19.3 20.7
Thiamin pyrophosphate 94.1 ± 30.9 109.9 ± 36.9 149.7
Pyrimidine Moiety 94.2 ± 7.1 83.9 ± 32.4 32.1
Riboflavin 100.9 ± 1.8 NA 3.6
Pyridoxal 96.9 ± 8.3 NA 9.9
Biotin 92.4 ± 10.1 92.6 ± 38 5.8
Adenosylcobalamin 99.1 ± 4.7 100.6 ± 16.4 6.1
Cyanocobalamin 99.1 ± 7.2 96.0 ± 18.5 1.3
Hydroxycobalamin 91.8 ± 7.6 109.7 ± 27.1 2.5
Methylcobalamin 94.5 ± 14.2 91.1 ± 37.5 3.4
Methionine 88.6 ± 15.2 NA 7.2
27
to partition into the chloroform. The liquid phase extraction effectively reduced the matrix
suppression problem, allowing for clear quantification of our target analytes.
To our knowledge, there is no standard reference material for particulate B-vitamins in
bacterioplankton and/or phytoplankton. Therefore, full particulate B-vitamin extraction must be
inferred from determining that filters and media used do not have a B-vitamin blank, observing
complete cellular lysis, determining the effectiveness of our lysis solution in separating B-
vitamins from the cellular matrix, and testing that our extraction procedure does not degrade B-
vitamins. No B-vitamin blank was observed on the Supor filters, and no B-vitamins from the
media used were observed to adsorb onto the filters. Complete cellular lysis was observed via
epiflourescence microscopy. Our lysis solution is similar to previously published particulate
extraction solutions that have been determined to result in full extraction of metabolites from the
cellular matrix (Roberts et al. 2012). We tested the relative accuracy and precision of our
extraction procedure by adding known concentrations of each vitamin congener to samples
containing filters with a known amount of Synechococcus CC9311 cells, and by extracting as
described above. A parallel sample with only CC9311 filtered cells was also analyzed. Standard
recoveries were calculated by subtracting the observed CC9311 vitamin signal from the
standards + CC9311 signal. These values were compared to extractions of known concentrations
of vitamin standards without CC9311 cells, and no significant differences in recovery efficiency
were observed, indicating that the addition of cellular organic compounds does not interfere with
our analysis. Spike recoveries (listed in Table 2.2) were >88% for all analytes.
2.4.2 Dissolved Method Validation
In order to fully complement the newly developed particulate B-vitamin method
described above, we expanded the previously established dissolved B-vitamin method to include
28
TMP, TPP, HMP, AB
12
, HB
12
, and MB
12
. In order to test the recoveries of these additional
analytes, known concentrations of standards were added to vitamin free seawater and were
preconcentrated using C
18
resin at two pHs (6.5 and 2.0) as has been described previously
(Okbamichael and Sañudo-Wilhelmy 2004; Okbamichael and Sañudo-Wilhelmy 2005; Sañudo-
Wilhelmy et al. 2012). After LC/MS analysis, recoveries were determined to be >83% for all
newly added analytes (Table 2.2). We attempted to include the B
1
precursor thiazole in the
dissolved method, however we observed that it did not successfully bind to our preconcentration
resin.
2.4.3 Internal Standard
Two internal standards (IS) were used for sample quantification. Stable isotopically
labeled B
1
(
13
C,
15
N, mass shift +4, Sigma-Aldrich) was used to quantify aqueous phase eluting
analytes (B
1
, TMP, TPP, HMP, MET), and stable isotopically labeled B
2
(
13
C,
15
N, mass shift +6,
Sigma-Aldrich) was used to quantify organic phase eluting analytes (B
2
, B
6
. B
7
, AB
12
, CB
12
.
HB
12
, MB
12
). The relationship between each analyte and the IS used for quantification was
empirically determined by calculating a response factor (RF) for each of these molecules (Table
2.1, Figure 2.2) (Bohn and Walczyk 2004).
Figure 2.2 Response Factor (RF) is calculated using E1 to determine the relationship between known concentrations
of the Internal Standard (C
IS
) and target analytes (C
VIT
), with the observed instrumental response (curve area) of the
Internal Standard (A
IS
) and the target analyte (A
VIT
). The concentration of the target analyte in a sample is then
calculated using E2 where the RF is used to correct for the differing instrumental response between the Internal Standard
and the target analyte.
E1: !"=
!
!"
!
!"
!
!"#
!
!"#
E2: !
!"#
=
!" !
!"
!
!"#
!
!"
29
Quantification using the IS and RF values was validated by analyzing known concentrations
of standards dissolved in LC/MS-grade water, and comparing concentrations calculated using the
IS/RF versus external calibration curves. During sample analysis external calibration curves
containing both target analytes and internal standards were run daily and used to verify the RF
and to correct for inter-day instrument variability/drift. IS intraday variability ranged between 6-
25% and 4-14% for B
1
-IS and B
2
-IS respectively; interday variability was 15% and 9% for B
1
-IS
and B
2
-IS respectively. The IS increased our analytical precision, and allowed for the correction
of matrix suppression effects which are known to be common (Heal et al. 2014).
Discussion 2.5
Particulate B-vitamins were extracted from cultures of two species of marine bacteria of
differing metabolic lifestyles: Synechococcus strain CC9311 (photoautotroph), Vibrio strain
AND4 (heterotroph). The normalized concentration (mole analyte mole POC
-1
) observed in
CC9311 for B
1
, B
7
, and CB
12
were 3.9x10
-5
±7.8x10
-6
and 9.9x10
-6
±8.1x10
-6
, and 4.2x10
-
7
±2.9x10
-7
, respectively (Table 2.3, Figure 2.3). The concentrations of these compounds in
AND4 were lower than those measured in CC9311, with the differences of the means of B
1
, B
7
,
and CB
12
concentrations equaling 3.5x10
-5
, 7.4x10
-6
, and 3.6x10
-7
, respectively (Table 2.3,
Figure 2.3). Recent research has suggested that cyanobacteria, including CC9311, solely have the
ability to synthesize and utilize pseudocobalamins, which potentially makes our observed
presence of CB
12
within CC9311 anomalous (Helliwell et al. 2016; Tanioka et al. 2009).
However we do not believe that these findings run contrary to our observations as our cultures
were not tested for axenicity at the endpoint of our experiment, and contamination with
heterotrophic bacteria could have occurred. In addition, the ability of CC9311 to remodel
pseudocobalamin is not well understood. We hypothesize that the observed differences in the
30
particulate concentration of each vitamin among species, which can span several orders of
magnitude, could potentially be associated with differing metabolic rates or B-vitamin
requirements between the species. B-vitamins are intrinsically linked with metabolic processes,
so a more metabolically active organism may require higher concentrations of particulate B-
vitamins (Banerjee 1997; Begley et al. 1999; Sañudo-Wilhelmy et al. 2014; Waldrop et al. 2012).
However, we did not assess growth rate or end-point axenicity in our cultures, so we cannot
definitively test this hypothesis using our data. Further studies using cultures and tightly
constrained growth rates are necessary to elucidate particulate B-vitamin requirements.
Table 2.3 Particulate B-vitamin obtained from cultures and environmental samples collected at SPOT. Values were
normalized to POC (mole particulate vitamin/mole POC). Blank cells indicate that the analyte was not included in the
LC/MS analysis. Conversion factors of 20fgC/cell and 200fgC/cell were used for AND4 and CC9311 respectively. The
POC concentration was measured at SPOT (16.4µM).
mean sd mean sd mean sd
Thiamin 3.83E-06 1.63E-06 3.90E-05 7.81E-06 3.23E-05 1.37E-05
Thiamin monophosphate 6.47E-07 4.79E-07 1.06E-05 7.09E-06
Thiamin pyrophosphate 3.45E-07 1.72E-07 1.24E-06 1.26E-06
Pyrimidine Moiety 3.37E-05 1.98E-05
Riboflavin 2.21E-06 1.08E-06 8.84E-06 3.74E-06
Pyridoxal 1.23E-05 5.01E-06
Biotin 2.47E-06 1.36E-06 9.89E-06 8.18E-06 2.19E-06 8.32E-07
Adenosylcobalamin 1.08E-07 6.15E-08 4.24E-07 4.94E-07
Cyanocobalamin 5.62E-08 4.20E-08 4.18E-07 2.95E-07 1.11E-07 1.84E-07
Hydroxycobalamin 8.62E-08 6.54E-08 2.84E-06 1.21E-06
Methylcobalamin 2.77E-07 1.30E-07 2.30E-07 3.76E-07
Methionine 7.59E-05 3.56E-05 4.11E-06 1.68E-06
Vibrio str. AND4
Synechococcus
str. CC9311 SPOT Surface
31
Figure 2.3 Particulate B-vitamin concentrations, normalized to cellular carbon, from cultures of Synechococcus
str.CC9311 (green squares), Vibrio str. AND4 (orange circles), cultures of marine eukaryotic phytoplankton from
Carlucci and Bowes (1972) (pink triangles), and from the surface ocean at the San Pedro Ocean Timeseries (SPOT,
33°33’N, 118°24’W) collected in March 2015 (purple diamonds). Conversion factors of 20fgC/cell and 200fgC/cell were
used for AND4 and CC9311 respectively. POC was measured at SPOT (16.4µM).
In order to evaluate the particulate pool of B-vitamins in environmental samples, we
collected seawater from two contrasting ocean systems. Sampling occurred at SPOT and at an
oceanic station in the subtropical Eastern Atlantic Ocean (see Experimental Sample Collection
section). The observed particulate concentrations (pico mole vitamin per liter of seawater, pM) at
3m at SPOT were 481±204 B
1
, 156±103 TMP, 17.6±16.8 TPP, 40.8±14.8 HMP, 129±45.7 B
2
,
31.9±9.87 B
7
, 5.82±6.53 AB
12
, 1.54±2.46 CB
12
, 40.8±14.8 HB
12
, 3.14±5.02 MB
12
, and
62.1±30.1 Met. At the Atlantic Station, particulate concentrations (pM) in the mixed layer
ranged from 3.53-44.4 B
1
, 5.64-46.5 TMP, 1.13-7.85 TPP, 5.91-45.56 HMP, 1.68-8.55 B
7
, 0.52-
4.53 AB
12
, undetectable-1.21 CB
12
, 1.21-9.11 HB
12
, 0.19-2.46 MB
12
, and 1.97-16.11 Met (Figure
2.4, Table 2.4). SPOT was more eutrophic during our sampling with a mean observed POC
1x10
-8
1x10
-6
1x10
-4
B
1
TMP
TPP
HMP
B
2
B
6
B
7
MET
AB
12
CB
12
HB
12
MB
12
Analyte
mole Pvit/mole POC
CC9311
AND4
Carlucci and Bowes 1972
SPOT
32
concentration of 16.4µM, whereas POC was observed to range between 1-4.5µM throughout the
mixed layer at the Atlantic station (data not shown). Normalization using the POC data allowed
us to take into account the density of biomass at each location. The normalized particulate B-
vitamin stoichiometric molar ratios (mole particulate vitamin: mole particulate organic carbon)
observed at SPOT (3m) in March 2015 were: 3.2x10
-5
±1.4x10
-5
B
1
: 1.1x10
-5
±7.1x10
-6
TMP:
1.2x10
-6
±1.3x10
-6
TPP: 3.4x10
-5
±1.9x10
-5
HMP: 8.8x10
-6
±3.7x10
-6
B
2
: 2.2x10
-6
±8.3x10
-7
B
7
:
4.2x10
-7
±4.9x10
-7
AB
12
: 1.1x10
-7
±1.8x10
-7
CB
12
: 2.8x10
-6
±1.2x10
-6
HB
12
: 2.3x10
-7
±3.7x10
-7
MB
12
: 4.1x10
-6
±1.7x10
-6
Met: 1POC (Table 2.3, Figure 2.3). The observed particulate B-vitamin
to POC molar ratios observed at 3m in the Atlantic are 8.1x10
-6
±1.6x10
-6
B
1
: 6.2x10
-6
±5.9x10
-7
TMP: 1.8x10
-6
±2.3x10
-7
TPP: 1.3x10
-6
±4.2x10
-7
HMP: 4.2x10
-6
±3.3x10
-7
B
2
: 1.8x10
-6
±6.7x10
-7
B
7
: 1.8x10
-7
±1.4x10
-7
AB
12
: 1.4x10
-6
±2.0x10
-7
HB
12
: 5.2x10
-8
±3.8x10
-8
MB
12
: 6.8x10
-7
±1.6x10
-7
Met: 1POC. The observed particulate vitamin concentrations (pM), and the POC normalized
values (mole particulate vitamin: mole POC) at 3m in the Atlantic were lower by as much as two
orders of magnitude than those observed at 3m at SPOT for each analyte, likely because the
Atlantic station was much more oligotrophic than SPOT. It is important to note that since a
prefilter was used for sample collection, the particulate B-vitamin concentrations measured at
our study sites only represent the bulk community ranging from 0.2µm-10µm, which likely
excludes large eukaryotic phytoplankton. The observed concentrations of particulate B-vitamins
in a bulk community measurement are a function of both the size of the community (biomass
standing stock), and potentially the growth rate of the individual organisms in the community.
We hypothesize that the relatively oligotrophic conditions in the Atlantic (compared to the
upwelling dominated, blooming community at SPOT) should result in lower microbial growth
rates, and therefore likely lower particulate B-vitamin requirements.
33
Figure 2.4 Particulate (blue triangles) and dissolved (red circles) B-vitamin concentrations (pM) measured at four
depths in the mixed layer of the Eastern Atlantic Ocean (33°1’16”N, 12°14’28”W). The DCM (0.06µg ChlA/L) depth is
indicated in green. The nutricline, as defined by PO
4
-3
concentration, is indicated in purple.
Stoichiometric estimations showed that the B-vitamin concentrations measured in both
cultures and field-collected samples were, on average, between one and two orders of magnitude
higher than the values reported from the phytoplankton cultures analyzed by Carlucci and Bowes
(1972) (Figure 2.3). We believe that this difference could either be a result of the lack of
sensitivity of the bioassay used to determine vitamin concentration in their study and/or the
substantial B-vitamin degradation that we observed under their original high temperature and
acidity particulate extraction conditions (see Results section).
B
1
TMP TPP HMP B
7
MET AB
12
CB
12
HB
12
MB
12
0
50
100
150
200
0
50
100
150
200
0
200
400
600
0
30
60
90
0
25
50
75
100
125
0
10
20
30
40
50
0
200
400
600
800
0
5
10
15
20
0
2
4
6
0.00
0.25
0.50
0.75
1.00
1.25
0
5
10
0
5
10
15
20
Vitamin Concentration (pM)
Depth (m)
Dissolved Pool
Particulate Pool
34
Similarly, our measured particulate B-vitamin concentrations (normalized to POC) are,
on average, an order of magnitude higher than published vitamin B
1
and B
12
calculated cellular
quotas based on uptake rates (Droop 2007; Paerl et al. 2015; Tang et al. 2010). Cellular quotas
of B-vitamins (pmole B-vitamin cell
-1
) for individual strains of eukaryotic phytoplankton have
been previously estimated using differential growth rates in varying concentrations of B-vitamins
(Croft et al. 2005; Droop 2007; Paerl et al. 2015; Tang et al. 2010). These published per cell
values were normalized to C values using published conversion factors: 20fgC/cell bacteria,
200fgC/cell cyanobacteria, 138pgC/cell microplankton (Caron et al. 1999; Waterbury et al.
1986). The vitamin per carbon values from the published uptake studies (obtained using the
above conversion factors) were compared to the mean vitamin per carbon values from 3m at
SPOT and all depths at the Atlantic Station. The mean total thiamin (B
1
, TMP, TPP combined)
and total cobalamin (all congeners combined) concentrations (mole B-vitamin mole POC
-1
)
measured at SPOT and at the Atlantic station are 2.3x10
-5
±6.8x10
-6
and 2.2x10
-6
±7.7x10
-7
,
respectively (Table 2.3 and Table 2.4). In comparison, the mean estimated cellular quotas for
thiamin and cobalamin obtained from Tang et al. (2010) and Paerl et al. (2015) are 6.3x10
-
7
±8.7x10
-7
and 2.4x10
-8
±5.9x10
-8
(mole B-vitamin mole POC
-1
), respectively. The 1-3 orders of
magnitude difference between the calculated cellular quotas (based on vitamin uptake rates), and
our observed particulate concentrations seems to point to the possibility that complex
environmental samples consisting of mixed communities may contain individual members with
higher B-vitamin quotas than those previously studied in monocultures. Furthermore, these data
also suggest that B-vitamin uptake could be a poor indicator for particulate B-vitamin
concentrations in environmental samples as the cellular quotas of organisms that have B-vitamin
synthesis capacity are not constrained by B-vitamin uptake rates. Future studies coupling B-
35
vitamin uptake rates and direct particulate measurements will be necessary to fully understand
these relationships.
Table 2.4 Dissolved (A.) and particulate (B.) B-vitamin and their congener concentrations (pM) determined at a
station in the Eastern Atlantic Ocean (33°1’16”N, 12°14’28”W) in May of 2014.
The dissolved pool of B-vitamins was measured at the Atlantic Station in conjunction
with the particulate pool. The observed concentration range (all in pM) of B-vitamins in the
dissolved pool are: 33.4-457.1 B
1
, 9.8-89.2 TMP, 2.5-77.3 TPP, 2.5-28.3 HMP, 40.1-679.4 B
7
,
0.07-3.5 AB
12
, 0.09-0.8 CB
12
, 0.2-3.1 HB
12
, 0.4-12.1 MB
12
, and 0.1-2.1 Met (Table 2.4, Figure
2.4). These concentrations were decoupled from inorganic nutrient concentrations (e.g. PO
4
-3
).
A.
mean sd mean sd mean sd mean sd
Thiamin 182 23.2 457 118 33.4 1.67 223 54.3
Thiamin monophosphate 33.9 2.74 70.1 21.5 9.83 2.62 89.2 21.9
Thiamin pyrophosphate 2.51 1.91 59.9 4.18 77.3 44.7 38.9 3.11
Pyrimidine moiety 2.53 0.64 28.3 3.19 2.90 0.11 4.56 2.27
Biotin 679 136 629 168 40.1 10.5
Adenosylcobalamin 3.49 2.34 1.33 0.28 0.07 0.01 1.80 1.75
Cyanocobalamin 0.80 0.43 0.59 0.04 0.09 0.02 0.48 0.25
Hydroxycobalamin 3.13 0.15 1.97 0.71 0.15 0.13 2.49 0.87
Methylcobalamin 12.1 8.46 6.97 2.30 0.37 0.13 7.33 1.68
Methionine 1.07 0.02 2.12 0.07 0.00 1.61 0.58
B.
mean sd mean sd mean sd mean sd
Thiamin 36.0 7.19 44.4 8.36 27.6 1.97 3.54 1.24
Thiamin monophosphate 27.9 2.62 46.5 6.59 20.7 6.11 5.64 0.60
Thiamin pyrophosphate 7.85 1.01 1.13 6.46 1.20 0.30
Pyrimidine moiety 5.91 1.90 21.7 5.05 45.6 2.79 7.31 1.87
Biotin 8.22 3.00 8.56 2.42 8.35 1.32 1.68 0.29
Adenosylcobalamin 0.82 0.64 2.11 0.69 4.54 1.57 0.52 0.11
Cyanocobalamin 1.22 0.01 0.01
Hydroxycobalamin 6.38 0.89 9.11 4.07 3.39 1.10 1.21 0.13
Methylcobalamin 0.23 0.17 2.46 0.12 0.33 0.27 0.20 0.02
Methionine 3.06 0.71 6.36 1.73 16.1 3.00 1.97 1.55
Dissolved Fraction, pM
Particulate Fraction, pM
3m 50m 85m 200m
3m 50m 85m 200m
36
Vitamins in the dissolved pool are sourced from biogenic processes, therefore areas that contain
the most biochemically active organisms, and likely the highest particulate B-vitamin
concentrations, have potential to produce the highest dissolved vitamin concentrations. The
relationship between the dissolved and particulate pools varies between analyte, and sample
depth, which likely indicates rapid scavenging and release of these valuable compounds.
In order to evaluate the relative enrichment of either the particulate or the dissolved pool
of B-vitamins in the environment, ratios of particulate to dissolved concentration were calculated
(Figure 2.5, Table 2.5). Values greater than one indicate relative particulate pool enrichment,
while values less than one indicate a relative enrichment in the dissolved pool. Vitamins B
1
, B
7
,
and MB
12
were found to be enriched exclusively in the dissolved pool with ratio values ranging
from 0.02-0.83, 0.01-0.21, and 0.02-0.89 respectively (Figure 2.5, Table 2.5). Only the amino
acid methionine was found to be always relatively enriched in the particulate pool with ratio
values ranging from 1.23-238.8 (Figure 2.5, Table 2.5). Ratio values for all analytes peaked at
the DCM and decreased at the nutricline (Figure 2.5, Table 2.5). The maximum concentration of
biomass at the DCM is likely the cause of the relative enrichment of particulate B-vitamins at
this depth (Figure 2.5, Table 2.5). Increased biological activity at the DCM, driven by increased
nutrient, and potentially dissolved B-vitamin, availability driven by diffusive nutrient flux from
the nutricline (200m), likely creates this relative enrichment through the increase of the rates of
two processes: de novo B-vitamin synthesis and B-vitamin uptake from the dissolved pool. The
relative enrichment in the dissolved vitamins pool at the nutricline (200m) is likely a result of
remineralization of sinking particles, coupled with B-vitamin degradation within dead or dying
cells. Future research will be required to robustly define the relationship between these two
pools.
37
Figure 2.5 Particulate (blue triangles) and dissolved (red circles) B-vitamin concentrations (pM) measured at four
depths in the mixed layer of the Eastern Atlantic Ocean (33°1’16”N, 12°14’28”W). The DCM (0.06µg ChlA/L) depth is
indicated in green. The nutricline, as defined by PO
4
-3
concentration, is indicated iDissertation_CPS.docxn purple.
Table 2.5 Ratios between particulate and dissolved analytes (pM). Values greater than one indicate particulate pool
dominance, values less than one indicate dissolved pool dominance. Blank cells indicate that either the particulate or the
dissolved pools were not detectable. DCM is at 85 meters depth.
B
1
TMP TPP HMP B
7
MET AB
12
CB
12
HB
12
MB
12
0
50
100
150
200
0
50
100
150
200
0.1 10.0 0.1 10.0 0.1 10.0 0.1 10.0 0.1 10.0
Ratio Value
Depth (m)
3m 50m 85m 200m
Thiamin 0.20 0.10 0.83 0.02
Thiamin monophosphate 0.82 0.66 2.10 0.06
Thiamin pyrophosphate 3.12 0.02 0.08 0.03
Pyrimidine moiety 2.33 0.77 15.7 1.60
Biotin 0.01 0.01 0.21
Adenosylcobalamin 0.24 1.59 68.4 0.29
Cyanocobalamin 14.1 0.02
Hydroxycobalamin 2.04 4.61 22.5 0.49
Methylcobalamin 0.02 0.35 0.89 0.03
Methionine 2.86 3.00 239 1.23
Intracellular Pool: Dissolved Pool
38
In summary, we have developed a direct method for the determination of particulate and
dissolved B-vitamins in the ocean. The particulate protocol involves a cellular extraction from
filtered cells, followed by a liquid phase extraction to remove organic molecules that could
interfere with the analyses. The dissolved method uses a C
18
resin to preconcentrate B-vitamins
from seawater. Analysis for both methods is carried out using a gradient elution using a C
18
column, followed by direct detection using a LC/MS. This new analytical protocol is used to
quantify vitamins B
1
, B
2
, B
6
, B
7
, B
12
, and the amino acid methionine as well as their chemical
congeners. This method is sensitive and accurate, and enables simultaneous direct quantification
of particulate and dissolved B-vitamins in the ocean. These techniques were successfully applied
to cultured marine bacteria and environmental samples from an upwelling-dominated eutrophic
coastal region off the coast of California, and an oligotrophic oceanic region in the Atlantic
Ocean. These techniques represent a much needed methodological expansion which will enable
important future studies to determine the roles that B-vitamins play in determining the structure
and productivity of marine microbial communities.
39
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42
Chapter 3 The potential role of dissolved and particulate B-
vitamins and their congeners influencing microbial diversity
in the Mediterranean Sea.
By Christopher P. Suffridge
Coauthors: Laura Gómez Consarnau, Lynda Cutter, Javier Arístegui, Xose A. Alvarez-
Salgado, Josep M. Gasol, Sergio A. Sañudo-Wilhelmy
43
Abstract 3.1
Understanding the factors that control phytoplankton growth and community structure is
critical for the understanding of global carbon cycling. Since the early 20
th
century, it has been
known that B-vitamins play a major role in determining phytoplankton community growth,
production, and composition. Limited oceanic dissolved B-vitamin distributions indicate that
these important coenzymes are present at pico-molar levels, which are too low to support
maximal phytoplankton growth, and there are vast regions of the ocean where they are
undetectable. Despite their importance, particulate B-vitamin quotas in field microbial
populations are unknown, as are the distributions of important biochemical B-vitamin congeners.
Here we report the B-vitamin concentrations measured in both the particulate and dissolved
fractions as well as measurements of multiple biochemically relevant B-vitamin congeners (B
1
,
TMP, TPP, HMP, biotin (B
7
), AB
12
, CB
12
, MB
12
, HB
12
, and the amino acid methionine). We
establish their spatial distributions spanning distinct microbiological and oceanographic regimes
in the Mediterranean Sea and the eastern Atlantic Ocean that show that all congeners are present
both dissolved in seawater and in suspended particles. We observe that B-vitamins co-occur in
biogeographic patches separated by large regions where B-vitamins are absent. Additionally, B-
vitamin quotas are observed to be highest at depths where Chl-a and bacterial activity are the
greatest, indicating that microbial B-vitamin requirements are related to growth rate. Finally,
linear regression models demonstrate strong, statistically significant, correlations between the
concentrations of dissolved B-vitamins and the abundances of microbes with obligate B-vitamin
requirements. These findings represent a substantial advancement in our understanding of the
ways that B-vitamins influence marine microbial ecology, and suggest that B-vitamin availability
has the ability to shape the microbial community structure, which could impact the strength of
the biological carbon pump.
44
Introduction 3.2
B-vitamins are essential coenzymes required in central metabolic pathways (Berg et al.
2007; Monteverde et al. 2017; Sañudo-Wilhelmy et al. 2014). It has been repeatedly
demonstrated, since the 1930s, that B-vitamins can influence the structure of phytoplankton
communities in marine systems (Carlucci and Silberna 1969; Croft et al. 2005; Croft et al. 2006;
Droop 1957; Droop 1962; Haines and Guillard 1974; Provasoli 1963; Provasoli and Carlucci
1974; Sañudo-Wilhelmy et al. 2014; Swift 1980). Recently, genomic data has demonstrated that
auxotrophy (obligate requirement) is substantially prevalent in marine systems, with a majority
of marine bacterioplankton species requiring the uptake of at least one B-vitamin (Bertrand and
Allen 2012; Bertrand et al. 2012b; Bertrand et al. 2007; Carini et al. 2014; Croft et al. 2006;
Gobler et al. 2007; Koch et al. 2012; Koch et al. 2011; Koch et al. 2013; Paerl et al. 2015; Paerl
et al. 2016; Panzeca et al. 2008; Panzeca et al. 2006; Sañudo-Wilhelmy et al. 2014; Tang et al.
2010). This includes the abundant, globally-distributed SAR11 clade of alphaproteobacteria,
which is an auxotroph for multiple B-vitamins and some of their intermediate precursors (Carini
et al. 2014; Giovannoni et al. 2005). In contrast, a small subset of heterotrophic bacteria and
cyanobacteria, such as Prochlorococcus and Synechococcus, are known to synthesize B-vitamins
(Sañudo-Wilhelmy et al. 2014). B-vitamins can influence different members of both eukaryotic
and prokaryotic microbial community via a complex ecological interplay between producers and
auxotrophs. However, most of the field studies to date have only focused on their impact on
eukaryotic phytoplankton (Bertrand and Allen 2012; Bertrand et al. 2012a; Bertrand et al. 2007;
Gobler et al. 2007; Koch et al. 2011; Paerl et al. 2015; Paerl et al. 2016; Panzeca et al. 2008;
Panzeca et al. 2006). One of the questions that remains unanswered is whether or not the
geographical distribution of different members of the plankton is related to the availability of
these exogenous metabolites.
45
Some B-vitamins are metabolically active in several different chemical forms. For
example, cobalamin congeners (vitamin B
12
) include the active coenzymes methylcobalamin
(MB
12
) and adenosylcobalamin (AB
12
) as well as the inactive/transitive congeners
cyanocobalamin (CB
12
) and hydroxycobalamin (HB
12
) (Banerjee 1997; Banerjee and Ragsdale
2003; Brown 2005; Koutmos et al. 2009; Quadros 2010; Watanabe and Nakano 1991). Thiamin
(vitamin B
1
) can be found as free thiamin and as its phosphorylated forms; thiamin
monophosphate (TMP), and the active form, thiamin pyrophosphate (TPP) (Begley 1996; Begley
et al. 1999). In addition, the thiamin molecule is itself composed of two motifs: a thiazole and a
pyrimidine moiety (HMP) that are separately synthesized before being ligated together in the
final biosynthetic steps (Begley 1996; Edwards et al. 2017; Kraft and Angert 2017). Laboratory
results have demonstrated that some organisms require the intact thiamin molecule while in
others, vitamin B
1
limitation could be alleviated by the uptake of either one of the two moieties
or both (Carini et al. 2014). Recent studies have also shown that some phytoplankton can fulfill
their thiamin requirements with the uptake of the phosphorylated forms of thiamin (Paerl et al.
2015; Paerl et al. 2016). Therefore, the availability of the different B-vitamin variants in the
environment could potentially benefit particular members of the microbial community over
others. However, the environmental presence, and therefore relevance, of these compounds is
still unclear. The concentration of some of vitamin congeners in seawater have only been
measured in a few occasions (Carini et al. 2014; Heal et al. 2014; Sañudo-Wilhelmy et al. 2012;
Suárez-Suárez et al. 2011; Suffridge et al. 2017), and their spatial and temporal gradients as well
as their impact on the microbial-phytoplankton diversity in the field are to date unknown.
Over the last few decades, ocean ecosystem modelers have started to include important
environmental parameters to build more accurate ecosystem dynamics models. However, these
46
models have not yet included the B-vitamins despite their well-known effects on community
composition. This exclusion is due to the lack of data on cellular B-vitamin quotas, which would
be required for model parameterization, and have only been obtained in a few species of
eukaryotic phytoplankton (Gobler et al. 2007; Koch et al. 2011; Tang et al. 2010). Furthermore,
the currently available intracellular quotas were estimated using dose-response methods that do
not necessarily represent the environmental variability observed in marine systems (Smirnov and
Revkova 2002). Therefore, actual B-vitamin quotas measured in natural microbial communities
are still unknown. One way to overcome this limitation is to simultaneous determine the
concentrations of B-vitamins in the extracellular (dissolved) and intracellular (particulate) pools
in the marine environment. These data would also help to better understand the mechanism
influencing the cycling of those growth factors between those two vitamins pools.
In this study we measured, for the first time, several chemical forms of B-vitamins (B
1
,
TMP, TPP, HMP, biotin (B
7
), AB
12
, CB
12
, MB
12
, HB
12
) and the amino acid methionine in both
the dissolved pool and in suspended particles collected in contrasting biological regimes
throughout the Mediterranean Sea and the eastern Atlantic ocean. Using those measurements we
establish B-vitamin spatial distributions and identify the processes influencing their geographical
trends. Particulate concentrations provide directly measured B-vitamin quotas that include
prokaryotic dominated oligotrophic microbial communities, which have been conspicuously
absent from the literature. Concentrations of dissolved B-vitamin and their chemical congeners
were used identify areas of potential B-vitamin limitation within the Mediterranean Sea.
47
Methods 3.3
3.3.1 Cruise Track
Samples were collected on an oceanographic cruise aboard the Spanish research vessel R/V
Sarmiento de Gamboa which began on April 26
th
, 2014 and ended on May 30
th
, 2014. The
cruise originated near Cyprus, continued westward through Mediterranean Sea and ended near
the Canary Islands (Figure 3.1). Sampling was conducted at twenty nine stations. During the
cruise samples for dissolved B-vitamins were collected at fourteen stations, and particulate B-
vitamins were collected at nine of those stations (Figure 3.1). Samples were collected at an
average of four depths in the photic zone (upper 300m) including at the surface and at the deep
chlorophyll max. Dissolved nutrients (nitrate, phosphate, silica), particulate organic carbon
(POC), particulate organic nitrogen (PON), and chlorophyll A (Chl-a) were measured at all
stations and depths. In addition, flow cytometry was conducted at each station and depth.
Figure 3.1 Mediterranean Sea-Atlantic Ocean Cruise Transect. The cruise occurred in the spring of 2014. It
originated at Station 1 near Cyprus and concluded at station 29 in the eastern Atlantic Ocean. Stations labeled in green
only have dissolved vitamin measurements. Stations labeled in red have paired dissolved and particulate vitamin
measurements. Regional boxes are based on biogeography.
1 2
3
5 4
8
7
9
6
11
10
12
17
16
15
14
13
21
20
19
18
27
26
25 23
22
28
29
24
Eastern Region
Western Region Atlantic
FIGURE 2.S1
48
3.3.2 B-vitamin Sample Collection
Samples for B-vitamins were collected as has previously been described (Suffridge et al.
2017). At least three liters of water was collected from each depth and was filtered using a
methanol cleaned, seawater rinsed peristaltic filtration apparatus. A 10µm mesh prefilter was
used. The particulates were collected on a 0.2µm pore size, 47mm diameter Supor filter (Pall
Biosciences). Particulate samples were transferred into cryovials and frozen at -80C until
analysis. The filtrate was collected in methanol cleaned amber HDPE bottles for dissolved-pool
analysis. Dissolved samples were frozen at -20C until analysis.
3.3.3 B-vitamin Analysis
B-vitamin samples were analyzed as has been previously described (Suffridge et al. 2017).
Dissolved samples were preconcentrated by passing the sample over a C
18
resin at two pHs (6.5
and 2.0), followed by elution into 12ml of methanol. A nitrogen dryer was used to evaporate the
samples to 250µl, providing a concentration factor of six orders of magnitude. Particulate B-
vitamins were extracted from the filters by bead beating in 3ml of acidic methanol lysis solution
followed by a chloroform liquid phase extraction to reduce signal suppression (Suffridge et al.
2017). Analysis of both dissolved and particulate B-vitamin samples was conducted using a
Thermo Scientific Quantum Access electrospray ionization triple quadrupole mass spectrometer,
coupled to a Thermo Scientific Accela High Speed Liquid Chromatography (LC/MS) system
(Suffridge et al. 2017). The LC system used a stable- bond C
18
reversed-phase column
(Discovery HS C
18
10 cm × 2.1mm, 5µm column, Supelco Analytical), with a methanol:water
gradient program (Suffridge et al. 2017). Quantification was conducted using two stable
isotopically labeled internal standards (thiamin and riboflavin) (Suffridge et al. 2017).
49
3.3.4 Biomass Parameters Sample Collection and Analysis
Dissolved nitrate, phosphate and silica were collected in acid washed HDPE bottles and
analyzed using standard protocols (Knap et al. 1996). Particulate organic carbon and nitrogen
samples were filtered on precombusted GF/F filters under low vacuum pressure, desiccated at
60°C, and analyzed using a CHN elemental analyzer (Knap et al., 1996). Chl-a samples were
collected on GF/F filters under low vacuum pressure, and the pigment was extracted using
acetone. Concentrations were determined fluorometrically using a Turner Designs 10AU
flourometer to analyze acetone extracted samples (Knap et al. 1996). Samples for flow cytometry
were collected, fixed with 2% formalin, and frozen at -80°C. Abundances of autotrophic
picoplankton (Prochlorococcus, Synechococcus, and picoeukaryotes) and heterotrophic
prokaryotes were determined using a Becton–Dickinson FACScalibur Flow Cytometer (Gasol
and Del Giorgio 2000).
3.3.5 N* Calculation
We calculated tracer N* as a means to place our dissolved vitamin concentrations in the
context of nutrient balance. N* values were calculated using the observed nitrate and phosphate
concentrations, and using a correction constant (2.9) that had previously been determined for the
Mediterranean sea (Deutsch et al. 2001; Gruber and Sarmiento 1997; Martiny et al. 2013).
Values ranged from 1.9 to 7.4 indicating extreme phosphorus limitation was present in large
portions of the study area, which has previously been reported (Deutsch et al. 2001; Gruber and
Sarmiento 1997; Martiny et al. 2013; Sarmiento and Gruber 2006).
50
3.3.6 Statistical Analysis
All data was analyzed, and all statistical analysis was conducted using the R software
environment. All figures were created using the Ggplot2 software package for R (R Core Team
2015; Wickham 2009).
Results and Discussion 3.4
Nutrient concentrations measured along the Mediterranean Sea-Atlantic Ocean transect
were low, with phosphate concentrations ranging from below detection to 0.1µM in the upper
100 meters (Figure 3.2). The exception to this trend was at the Gibraltar Strait station (station
24), where an outcropping of nutrient rich water (phosphate concentrations >0.2µM) reached to
just below the surface (Figure 3.2). The oligotrophic conditions that we observed during our
cruise are characteristic of the Mediterranean Sea, and the relatively high nutrient levels at the
Gibraltar Strait are caused by the advection of Atlantic surface water into the Alboran Sea in the
western Mediterranean (Bergamasco and Malanotte-Rizzoli 2010; Krom et al. 2010; Siokou-
Frangou et al. 2010; Tanaka et al. 2007; Thingstad et al. 2005). While nutrient concentrations
were relatively low, clear biogeographic trends were discernable, and a deep chlorophyll
maximum (DCM) was clearly defined throughout the transect (Figure 3.2). The DCM was
deepest in the Eastern basin (east of the strait of Sicily, stations 1-12) ranging between 130m-
100m, and shallowest in the western basin (Strait of Sicily to Strait of Gibraltar; stations 14-24)
where it was centered around 60m. In the eastern Atlantic Ocean the DCM deepened with
increasing distance from the coast, with an average depth of 80 m. A strongly defined DCM is
characteristic of the Mediterranean Sea, and the observed DCM depths are within the ranges that
have been previously reported (Siokou-Frangou et al. 2010). Regardless of region, the location of
the DCM closely matches the zone of optimal Redfield N:P ratio (16:1) as defined by the tracer
51
N* calculated for the Mediterranean Sea (Figure 3.2)(Gruber and Sarmiento 1997; Martiny et al.
2013; Moore et al. 2013).
Our data indicate that the primary contributors to the observed chlorophyll signal were
the picophytoplankton, which is consistent with what would be expected in an oligotrophic
system such as the Mediterranean Sea during our sampling (Amorim et al. 2016; Chisholm et al.
1988; Schauer et al. 2003; Siokou-Frangou et al. 2010; Tanaka et al. 2007). Prochlorococcus
was the dominant photoautotroph in the Eastern Basin and the Atlantic Ocean with abundances
reaching 1.1x10
6
and 1.3x10
5
cells/ml respectively (Figure 3.2). In the Western Basin, although
nutrient concentrations were similar to the rest of the transect, Prochlorococcus was nearly
absent (only present at two locations) and was replaced by Synechococcus with concentrations
reaching 7x10
4
cells/ml (Figure 3.2). The cyanobacteria are known to be dominant in the both
the upper waters and at the DCM in the Mediterranean Sea for the majority of the year and they
are primary contributors the observed Chl-a signal (Casotti et al. 2003; Marty and Chiavérini
2010; Siokou-Frangou et al. 2010; Tanaka et al. 2007). The cyanobacteria abundances reported
here are within the ranges characteristic for this region (Chisholm et al. 1988; Schauer et al.
2003; Tanaka et al. 2007). Synechococcus, is the primary phototroph in upper layers of the
Mediterranean Sea (Figure 3.2). This is likely because of its ability to efficiently photosynesize
at nutrient levels found in the western basin (Marty and Chiavérini 2010; Siokou-Frangou et al.
2010). Prochlorococcus reaches maximal abundances at the bottom of the photic zone near the
DCM with highest abundances in the Eastern Region (Figure 3.2). It has been previously
observed that the low light adapted Prochlorococcus ecotype is responsible for this sharp peak in
abundance demonstrating clear niche partitioning between groups of picoplankton (Moore et al.
1998; Siokou-Frangou et al. 2010).
52
Figure 3.2 Distributions of dissolved B-vitamins, nutrients, and biomass parameters. Concentrations total dissolved
thiamin, dissolved B
7
, and total dissolved cobalamin (pM) are plotted as a function of depth and longitude. Additionally
bacterial abundance (cells/ml), relative abundance of LNA cells (%), Synechococcus abundance (cells/ml),
Prochlorococcus abundance (cells/ml), Chlorophyll A concentration (pM), and dissolved phosphate concentration (µM)
are also plotted as a function of depth and longitude. N* value contours are overlaid on panels except phosphate where
the phosphate contours are plotted. The vertical gray line is at the Gibraltar Strait and the vertical blue line is at the
Sicily Strait.
Bacterial
Abundance
(cells/ml)
Relative
Abundance
LNA cells
(%)
Synechococcus
Abundance
(cells/ml)
Prochlorococcus
Abundance
(cells/ml)
Chlorophyll A
(pM)
Phosphate
( M)
80%
50%
30%
tB
1
(pM)
B
7
(pM)
tB
12
(pM)
53
We also observed a regional biological differentiation in the distribution of heterotrophic
bacteria with consistently higher abundances present in the western basin (5x10
5
-1.5x10
6
cells/ml), and lower abundances in the eastern basin (1x10
2
-5x10
5
cells/ml) (Figure 3.2). Similar
regional heterotrophic bacterial distributions have been previously reported (Pinhassi et al. 2006;
Siokou-Frangou et al. 2010). Copiotrophic bacterial groups Rhodobacterales, Flavobacteria, and
Bacteroidetes are known to be the primary groups of bacteria present and are responsible for the
majority of bacterial activity during the spring in the western region of the Mediterranean
(Pinhassi et al. 2006; Vila-Costa et al. 2012). These groups of bacterioplankton are known to
contain species that are B-vitamin auxotrophs (Sañudo-Wilhelmy et al. 2014). Bacterial
abundances were lowest in the Eastern Region, and this has been attributed to the extreme
phosphorus stress found in this region, although the importance of colimitaiton by coenzymes
has not been addressed (Siokou-Frangou et al. 2010). This region had the highest percent of low
nucleic acid cells (LNA), which have been determined to be primarily composed of cells from
SAR11 and SAR86 clades, although SAR11 has been shown to be the most abundant taxon in
the season that we sampled (Figure 3.2)(Gasol et al. 1999; Vila-Costa et al. 2012; Wang et al.
2009). SAR11 is known to require HMP, B
7
, and methionine (reduced sulfur source) (Carini
2013). Genomic and metagenomic studies of the uncultured SAR86 clade of
gammaproteobacteria indicate that is a B
1
, B
7
, methionine (protein synthesis), and B
12
auxotroph
(Dupont et al. 2012). It has been experimentally demonstrated that SAR11 does not respond to
phosphorus additions in the Eastern region of the Mediterranean despite the extreme nutrient
stress, suggesting that B-vitamin auxotrophy might be the driver of regional differential
abundances (Sebastian and Gasol 2013).
54
3.4.1 Dissolved B-Vitamin Concentrations
Total dissolved B-vitamin concentrations show a strong spatial pattern, and in large areas of the
Mediterranean-Atlantic transect, B-vitamins levels were found to be below our detection limits
(Figure 3.2; Table 3.1). The strong spatial variability in B-vitamin concentrations found during
our cruise has been reported in other regions of the world ocean suggesting that areas where B-
vitamins are undetectable are ubiquitous in the ocean (Sañudo-Wilhelmy et al. 2012). Dissolved
B-vitamins co-occurred in geographically distinct patches where their concentrations were an
order of magnitude greater than the area surrounding the patch (Figure 3.2). These vitamin-
enriched areas (on the eastern margin of the Ionian Basin, the North Balearic Front, the Algero-
Provencal Basin, Gibraltar Strait, and Atlantic Ocean) (Figure 3.1 and Figure 3.2) were generally
found in zones with slightly negative N* values, suggesting that P limitation was slightly
alleviated in those areas (Figure 3.2). Additionally, the location of the vitamins-enriched zones
are consistent with a detailed K-means cluster analysis of ten years of SeaWifs satellite imagery
that identifies seven separate biogeographic regimes in the Mediterranean Sea (D'ortenzio and
Ribera D'alcalà 2009). The patch in the Ionian Sea (stations 6 and 9) consists of two stations that
are in the approximate location of the permanent cyclonic Northeast Ioninan Sea and Cretan
Gyres, which are known to induce localized vertical transport of nutrients, and therefore
localized increases in biomass (Malanotte-Rizzoli et al. 1997; Siokou-Frangou et al. 2010). The
North Balearic Front (station 17) is characterized as one of the few regions in Mediterranean Sea
where pronounced phytoplankton blooms occur (Bergamasco and Malanotte-Rizzoli 2010;
D'ortenzio and Ribera D'alcalà 2009; Siokou-Frangou et al. 2010). The Algero-Provencal Basin
(stations 20 and 22) is influenced by aeolian dust inputs, and well-defined mesoscale instabilities
caused by the interaction of Modified Atlantic Water and Mediterranean Water. It is well known
that the interaction of those two water masses induce localized fertilization events increasing
55
biological activity (Morán et al. 2001; Siokou-Frangou et al. 2010). Gibraltar is a shallow strait
where water flows in from the Atlantic causing a well documented increase in biological
productivity, and linking the observed B-vitamin patch with the observed patch in the Atlantic
(station 25) (Bergamasco and Malanotte-Rizzoli 2010; D'ortenzio and Ribera D'alcalà 2009;
Siokou-Frangou et al. 2010). Taken together these lines of evidence point to mesoscale
oceanographic features that control the microbial community and thus the distribution of total
dissolved B-vitamins.
56
Figure 3.3 Particulate B-vitamin depth profiles. Particulate B-vitamin concentrations (pM) plotted as a function of
depth for all analytes at all stations. The color of the profiles indicate their regional location with profiles from the
eastern region in orange hues, profiles from the western region in blue hues, and profiles from the Atlantic Ocean in
green hues.
0
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Dissolved B-Vitamin Concentration (pM)
25
27
28
29
B
1
TMP TPP HMP MET B
7
AB
12
CB
12
HB
12
MB
12
East Region West Region Atlantic Ocean
57
Station Depth (m) Latitude Longitude Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D.
2 3 34.073 31.413 18.0 6.16 0.274 5.91 3.60 4.30 1.83 133 31.0 0.710 0.210 0.822 0.114 0.0189 0.00353 0.107 0.0367 1.19 0.180
2 25 34.073 31.413 1.03 0.169 0.624 0.00609 0.0819 0.875 0.162 211 73.0 1.53 0.233 0.152 0.135 0.799 0.0961 2.44 1.66
2 130 34.073 31.413 5.52 1.61 2.60 1.38 10.2 5.47 2.27 0.296 1040 459 0.193 0.0230 2.33 1.38 1.08 0.722 1.53 0.740 8.82 3.57
2 200 34.073 31.413 479 43.5 74.2 16.3 8.69 2.44 5.25 969 213 2.78 0.234 7.86 4.74 0.870 0.349 3.31 1.11 12.2 2.09
4 3 34.073 27.226 17.8 2.06 4.70 1.10 1.34 1.93 0.813 115 34.7 0.0340 0.0130 0.318 0.0413 0.0909 0.0215 0.427 0.0639 0.915 0.112
4 50 34.073 27.226 16.8 2.93 3.72 0.591 0.463 0.309 0.159 0.189 0.103 0.0552 0.0832 0.00515 0.100 0.0376 0.642 0.0812
4 118 34.073 27.226 14.7 3.76 2.00 0.431 1.43 0.167 0.113 0.0126 0.0544 0.00642 0.0810 0.0187 0.414 0.136
4 200 34.073 27.226 13.0 1.19 3.85 1.08 0.407 0.117 0.201 0.0388 8.65 0.00798 0.00309 0.146 0.0642 0.0253 0.00278 0.0574 0.0226 0.813 0.0181
6 3 34.073 23.208 23.9 6.44 3.18 1.20 1.65 0.345 3.05 0.474 28.8 14.8 0.166 0.055 0.0294 0.0154 0.197 0.0613 0.533 0.0674
6 50 34.073 23.208 253 42.4 57.8 16.5 28.6 1.68 0.760 871 218 1.04 1.50 0.509 0.672 0.389 1.52 0.337 7.02 1.18
6 90 34.073 23.208 16.9 6.08 3.02 0.710 0.394 0.121 0.0267 0.0684 0.0149 0.0935 0.0359 0.161 0.055 0.616 0.161
6 200 34.073 23.208 8.29 1.15 1.22 0.253 0.0600 0.118 0.0258 0.0414 0.00895 0.00690 0.0173 0.00563 0.0705 0.0147 0.352 0.115
9 3 37.715 19.772 184 6.23 45.5 3.61 13.0 6.54 6.55 1.13 1950 237 0.451 0.209 0.468 0.0429 0.430 0.185 0.966 0.159 2.43 0.104
9 50 37.715 19.772 104 7.69 26.0 2.99 84.5 5.83 6.70 0.765 201 50.2 0.298 0.176 1.44 0.503 0.0845 0.0274 0.530 0.247 2.89 0.976
9 95 37.715 19.772 55.6 12.2 12.5 4.26 1.83 1.67 2.20 143 39.4 0.384 0.212 0.0470 0.0323 0.177 0.0635 0.650 0.258
9 200 37.715 19.772 1.07 0.385 1.05 187 50.2 0.126 1.22 0.303 0.237 0.0323 1.23 0.731 2.88 0.875
12 3 36.000 17.417 13.2 1.05 2.76 0.669 0.630 0.377 0.299 70.6 25.3 0.0245 0.0225 0.0656 0.0201 0.0935 0.00066 0.441 0.109
12 50 36.000 17.417 12.1 1.24 3.18 1.00 0.805 0.457 0.210 0.0519 140 22.4 0.0418 0.0189 0.0781 0.0238 0.125 0.0232 0.618 0.176
12 120 36.000 17.417 13.9 2.09 3.61 0.756 0.351 0.288 0.0567 0.0285 0.0739 0.0195 0.0461 0.00537 0.177 0.0990 0.695 0.326
12 200 36.000 17.417 25.2 4.72 5.70 3.90 24.5 5.81 7.10 7.47 10.9 0.157 0.496 0.131 0.126 0.0142 0.00186 0.0438 0.00541
14 3 37.845 11.419 0.485 0.092 2.12 1.89 0.537 0.0901 1.27 0.677 155 71.3 0.376 0.0255 0.0811 0.0221 0.362 0.159 1.28 0.308
14 40 37.845 11.419 2.39 0.209 0.521 0.425 196 43.3 0.522 0.580 0.180 0.0851 0.0319 0.363 0.192 1.47 0.190
14 300 37.845 11.419 36.0 1.11 5.44 0.342 0.277 0.0726 1.62 304 50.6 0.209 0.0979 0.484 0.242 0.0911 0.0355 0.256 0.0849 1.51 0.844
17 3 41.489 7.492 1.79 0.224 0.222 0.170 2.16 0.645 189 52.8 0.516 0.167 0.280 0.0447 0.0731 0.0357 0.249 0.0808 1.40 0.420
17 60 41.489 7.492 3.84 0.760 0.794 0.236 1.18 0.389 626 191 0.259 0.0768 0.267 0.0617 0.0339 0.0167 0.0691 0.0319 0.601 0.203
17 300 41.489 7.492 27.8 3.72 4.23 0.189 1.63 0.640 72.0 12.1 0.187 0.0242 0.0066 0.0188 0.0003 0.0559 0.0122 0.418 0.111
20 3 38.413 5.215 71.6 7.93 16.7 3.62 1.83 10.5 3.90 142 39.5 1.05 1.44 0.411 0.0507 0.0787 0.0176 0.140 0.0239 1.43 0.253
20 25 38.413 5.215 1.09 3.73 0.777 156 28.6 0.588 0.120 1.66 0.0711 0.0994 0.0322 1.61 0.906
20 65 38.413 5.215 886 233 195 46.1 183 89.4 189 75.8 1970 515 1.23 1.06 6.42 4.32 2.46 0.650 3.15 0.487 13.9 2.59
20 100 38.413 5.215 41.1 6.07 10.2 2.61 2.00 1.56 17.8 6.49 49.2 13.3 0.136 0.184 0.0114 0.0196 0.00878 0.0457 0.0123 0.589 0.0513
22 3 37.010 0.000 1.29 0.169 31.8 5.57 6.08 0.945 434 7.30 1.84 1.76 1.03 0.491 0.264 2.16 0.500 2.83 1.73
22 55 37.010 0.000 39.8 4.58 10.7 1.32 1.11 1.64 137 33.0 0.299 0.307 0.248 0.182 0.411 0.143 1.02 0.206
24 3 35.986 354.633 0.759 0.179 0.780 2.32 0.754 78.5 7.56 0.362 0.139 0.137 0.0130 0.0527 0.0135 0.160 0.0634 1.25 0.422
24 12 35.986 354.633 200 22.4 73.3 13.3 13.5 15.5 12.4 3.35 554 168 1.12 0.282 0.739 0.242 2.29 0.511 6.26 2.23
24 25 35.986 354.633 31.0 3.83 9.41 1.46 0.812 0.261 2.23 0.652 179 66.5 0.277 0.0707 0.0302 0.0122 0.114 0.00050 1.60 0.643
25 3 36.017 352.033 21.9 8.88 5.42 0.949 2.34 0.442 48.5 24.4 0.437 0.0912 0.0970 0.0329 0.219 0.0739 0.718 0.235
25 50 36.017 352.033 12.0 1.30 2.16 0.747 2.31 0.195 0.0665 0.0773 0.0168 0.0132 0.00285 0.193 0.0630 0.122 0.0300
25 75 36.017 352.033 21.8 4.53 4.78 1.64 0.357 0.0305 3.97 1.09 119 15.4 0.0287 0.00851 0.0933 0.0732 0.131 0.0780 0.204 0.0684 0.841 0.310
27 3 34.584 348.414 16.9 4.05 4.37 0.624 0.519 0.645 1.01 0.364 122 17.4 0.0490 0.0193 0.0181 0.0415 0.0338 0.153 0.0334 0.543 0.222
27 50 34.584 348.414 21.8 1.92 5.72 0.589 1.01 1.11 2.76 0.715 110 13.0 0.0714 0.150 0.0730 0.0656 0.00653 0.322 0.0289 1.11 0.462
27 80 34.584 348.414 28.2 2.77 10.5 1.39 1.13 0.0716 2.21 0.611 74.4 27.6 0.142 0.130 0.541 0.225 0.0455 0.00387 0.310 0.103 1.79 0.996
28 3 33.021 347.759 182 23.2 33.9 2.74 2.51 1.91 2.53 0.641 679 136 1.07 0.0183 3.49 2.34 0.805 0.431 3.13 0.153 12.1 8.46
28 50 33.021 347.759 457 118 70.1 21.5 59.9 4.18 28.3 3.19 629 168 2.12 1.33 0.278 0.595 0.0378 1.97 0.711 6.97 2.30
28 85 33.021 347.759 33.4 1.67 9.83 2.62 77.3 44.7 2.90 0.113 40.1 10.5 0.0674 0.00482 0.0663 0.0125 0.0865 0.0209 0.151 0.133 0.372 0.127
28 200 33.021 347.759 223 54.3 89.2 21.9 38.9 3.11 4.56 2.27 1.61 0.580 1.80 1.75 0.478 0.252 2.49 0.873 7.33 1.68
29 3 31.250 347.023 44.0 11.8 8.06 6.39 59.2 17.2 0.357 0.396 0.330 0.263 0.0491 0.0240 0.127 0.0284 0.150 0.0218
29 50 31.250 347.023 33.1 7.59 6.60 0.840 0.105 0.101 4.24 0.466 133 35.0 0.392 0.137 0.109 0.0290 0.629 0.263 1.01 0.320
29 70 31.250 347.023 100 34.8 26.1 7.00 2.77 0.381 463 93.2 0.396 0.0231 0.615 0.07502 0.345 0.0620 0.730 0.227 5.50 1.71
AB 12 CB 12 HB 12 MB 12 B 1 TMP TPP HMP B 7 Methionine
Table 3.1 Dissolved B-vitamin Concentrations (pM). Mean concentrations are provided for all analytes at all stations. Blank
cells indicate that the analyte was below detection limits at that location.
58
A comparison of the depth profiles obtained for nutrients and those for dissolved B-
vitamins measured along the Mediterranean-Atlantic Ocean transect show that both were
decoupled from each other (Figure 3.2 and Figure 3.3). Further evidence for this nutrient-vitamin
decoupling is provided by the absence of any correlations between the inorganic nutrients and
the organic coenzymes. This finding is not unique to the Mediterranean, as previous
measurements off the Southern California-Baja California coast in the Northeast Pacific margin
did not show any correlation between the vitamin and the inorganic nutrients (Sañudo-Wilhelmy
et al. 2012). In this study we observed maximal B-vitamin concentrations at, or above the DCM,
while the zones of maximal nutrients occurred below the mixed layer (Figure 3.2). This leads us
to conclude that different biological processes must be controlling nutrient and dissolved B-
vitamin distributions in the Mediterranean-Atlantic Ocean transect.
3.4.2 Dissolved B-Vitamin Congener Relative Abundance
This study provides the most comprehensive information yet available on the distribution
and description of the relative contribution to the total dissolved vitamin pool, of the congeners
of thiamin and cobalamin, in addition to the thiamin precursor HMP and the amino acid
methionine. All of these compounds were present in the transect, and their concentrations are
reported in Table 3.1, Figure 3.2, and Figure 3.3). The B
7
concentrations observed along our
cruise are the highest yet reported dissolved in seawater (8.6-1970 pM; Figure 3.2). Previous
LCMS based dissolved B
7
concentrations ranged from 1-500pM (Heal et al. 2014; Sañudo-
Wilhelmy et al. 2012). The concentrations reported here for all other congeners, except MB
12
,
are within the ranges that have previously been published using both LCMS and HPLC based
methods (Table 3.1) (Carini et al. 2014; Heal et al. 2014; Panzeca et al. 2009; Sañudo-Wilhelmy
et al. 2012; Suárez-Suárez et al. 2011; Suffridge et al. 2017). The reported concentrations of
59
MB
12
(0.04-13.8 pM) fall below the previously observed ranges from the NW Mediterranean
(33-83pM) and above those reported from Hood Canal (<0.1 pM) (Heal et al. 2014; Suárez-
Suárez et al. 2011). Our findings confirm the importance of measuring all four upper axial forms
of cobalamin, as exclusively measuring CB
12
(0.01-2.5 pM) underestimates the total available
cobalamin pool by an order of magnitude (tB
12
ranges from 0.12-24.1 pM). In contrast, our data
indicate that although exclusively measuring vitamin B
1
(0.5-885 pM) does underestimate the
total thiamin pool present (tB
1
ranges from 0.36-1200pM), the extent of the underestimation is
relatively minor. We observed that within the dissolved pool, the thiamin and the cobalamin
congeners all significantly and positively covary with each other (Table 3.2)(R Core Team
2015). These correlations are to be expected, as the cycling between the different chemical forms
of thiamin and cobalamin is closely coupled (Bridwell-Rabb and Drennan 2017; Fang et al.
2017; Helliwell et al. 2016; Paerl et al. 2015; Romine et al. 2017). The relative contribution of
the thiamin and cobalamin congeners to the total dissolved fraction was calculated at each station
and depth to determine the B-vitamin speciation across the transect (Figure 3.4). It is apparent
that for both the thiamins and the cobalamins, there is a dominant congener speciation pattern
that occurs over the majority of the transect. The interquartile ranges (IQR; the range of values
between the 25
th
and 75
th
percentile of the entire range) of thiamin relative abundances are 71-
83% B
1
, 13-20% TMP, and 1-10% TPP (Figure 3.4). Recent research seems to support these
observed ranges of vitamin B
1
congener relative abundance by providing evidence that natural
phosphatases in seawater have the ability to directly act on TMP and TPP cleaving it to B
1
(Paerl
et al. 2015; Paerl et al. 2016).
60
Table 3.2 Correlation matrix for concentrations (pM) of dissolved and particulate B-vitamins. Panels A and B (red)
are include dissolved pool data, panels C and D include particulate pool data. All values supplied are the Pearson
Correlation Coefficient. Only significant correlations (p-value<0.05) are included, therefore a blank indicates that no
significant correlation existed.
In the phosphorus-limited Mediterranean Sea it has been shown that alkaline phosphatases
are abundant, active, and an important part of the phosphorus acquisition strategy for the
microbial community (Wambeke et al. 2002). This is of substantial interest because TPP, which
is generally present in the smallest fraction, is the only active form of thiamin (Begley 1996).
Therefore, the thiamin congener in the greatest supply (B
1
) is not immediately bioactive, and
must be converted back to TPP via biological salvage pathways after uptake by the cell (Begley
1996). Interestingly, although the presence of alkaline phosphatases has been shown to be
ubiquitous in the Mediterranean Sea, there are several regions and depths were the fractionation
of TPP and TMP increase relative to B
1
(Figure 3.4; Wambeke 2002). The most dramatic
changes in the relative contribution of each of the thiamin congeners occurs at the surface and
mid depths in the Western Region of the Mediterranean and at the DCM at station 28 in the
Atlantic Ocean (Figure 3.4). The affinity of alkaline phosphatases for TPP and TMP has not been
established, and based off their picomolar concentration, it is likely that in these areas of higher
relative phosphorylated thiamin abundances, higher concentrations of more labile dissolved
organic phosphorus exist, competitively inhibiting the breakdown of TMP and TPP.
A. B.
tdB
1
B
1
TMP TPP HMP tdB
12
CB
12
HB
12
AB
12
MB
12
MET
tdB
1
0.99 0.97 0.83 0.84 tdB
12
0.90 0.95 0.93 0.99 0.71
B
1
0.95 0.77 0.81 CB
12
0.82 0.8 0.88 0.51
TMP 0.79 0.81 HB
12
0.84 0.82 0.73
TPP 0.83 AB
12
0.88 0.72
HMP MB
12
0.68
MET
C. D.
tpB
1
B
1
TMP TPP HMP tpB
12
CB
12
HB
12
AB
12
MB
12
MET
tpB
1
0.99 0.97 0.61 0.76 tpB
12
0.97 0.90 0.71 0.60
B
1
0.94 0.60 0.73 CB
12
TMP 0.80 HB
12
0.77 0.54 0.42
TPP AB
12
0.77 0.80
HMP MB
12
0.72
MET
61
Figure 3.4 Relative abundances of dissolved thiamin and cobalamin congeners. Relative abundances were calculated
as percent of the total dissolved thiamin and cobalamin pools respectively. Samples were binned into four depth
categories at each station: Surface (3m), Mid (3m-DCM), DCM, and Deep (DCM-300m). Some stations did not have a
sample in one bin, but there were no stations with two samples in one bin. The vertical gray line is at the Gibraltar Strait
and the vertical blue line is at the Sicily Strait.
Similar to the thiamin congeners, the dissolved cobalamins have a consistent speciation pattern
where MB
12
is the dominant congener at all but four locations. The IQR of the relative
abundance of MB
12
is 52-70% of the total dissolved cobalamin pool (Figure 3.4). CB
12
had the
lowest IQR of relative abundance ranging from 3-7% (Figure 3.4). AB
12
and HB
12
had
overlapping IQRs of relative abundances ranging from 10-23% and 11-19% respectively (Figure
3.4). The observed CB
12
concentrations could be a result of cellular excretion, as it has been
0
25
50
75
100
0
25
50
75
100
0
25
50
75
100
0
25
50
75
100
29 28 27 25 24 22 20 17 14 12 9 6 4 2
Station
Relative Abundance (%)
Dissolved Congeners
29 28 27 25 24 22 20 17 14 12 9 6 4 2
Surface Mid DCM Deep
CB
12
HB
12
AB
12
MB
12
B
1
TMP
TPP
62
shown that some cyanobacteria, including Synechococcus, excrete CB
12
, and based on the
maximum reported cellular CB
12
excretion, and the observed maximum abundances of
Synechococcus found in this transect, excretion could contribute up to about 1 pM of tB
12
(Bonnet et al. 2010). In the regions where maximum densities of Synechococcus were observed,
the mean dissolved CB
12
concentration was 0.31 pM, which demonstrates that excretion could be
a primary contributor to the dissolved B-vitamin pool (Bonnet et al. 2010). The speciation
pattern of the cobalamins is consistent with the findings of a study conducted in the northwest
Mediterranean Sea (HPLC based) where the relative abundance of MB
12
was observed to be 80%
and the combined relative abundance of the other cobalamins was only 20% (Suárez-Suárez et
al. 2011). AB
12
, also an active form of cobalamin responsible for radical mediated reactions such
as ribonucleotide reduction, replaces MB
12
as the dominant species at three locations: at the
surface in the Atlantic Ocean (station 29), between the surface and the DCM in the Algero-
Provencal Basin, and below the DCM in the Ionian Sea (station 12). HB
12
, and inactive form of
cobalamin becomes dominant only once, between the surface and the DCM in the Atlantic
Ocean (station 25). There is not a shared biological or chemical feature at these locations, so the
cause of the switch in speciation is not clear. MB
12
is the active form of cobalamin that is used
by organisms for the synthesis of methionine through the cobalamin dependent methionine
synthase (Matthews et al. 2003). It is surprising that the most abundant form of cobalamin
measured in the Mediterranean is an active form given the widespread presence of cobalamin
auxotrophy amongst marine microbes (Croft et al. 2006; Droop 2007; Heal et al. 2014; Sañudo-
Wilhelmy et al. 2014). However, the methyl axial ligand is a weaker leaving group than
hydroxyl ligand and the adenosyl ligand in the aqueous phase, which increases MB
12
s relative
stability compared to HB
12
and AB
12
(Mebs et al. 2009; Swetik and Brown 1974).
63
3.4.3 Particulate and Dissolved B-Vitamin Pool Partitioning
For the first time we are able to compare the standing stocks of both the dissolved and
particulate partitions of B-vitamins from simultaneously collected samples; these relationships
can provide insight to the cycling of B-vitamins between these two pools along the
Mediterranean-Atlantic transect (Figure 3.5). B-vitamins are biogenic molecules that are
synthesized exclusively within cells (Berg et al. 2007). Therefore, marine organisms (found
within the particulate pool) are the ultimate source of all B-vitamins found in the dissolved
fraction. However, it is expected that changes in temporal and spatial patterns in the partitioning
between the two pools are due to a combination of different biological processes including
vitamin uptake and de novo synthesis, as well as vitamin excretion, viral lysis, protistian grazing,
etc. (Koch et al. 2012; Koch et al. 2011; Paerl et al. 2015; Tang et al. 2010). Although our
environmental data does not allow us to directly evaluate the processes contributing to the
observed particle-dissolved partitioning of B-vitamins along our cruise, our results do allow us to
evaluate the contribution of each fraction to the total vitamin pool, and correlate them to the
distribution of biomass.
64
Station
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Particulate B-Vitamins (pM)
tB
1
HMP B
7
MET tB
12
Surface Mid DCM
Figure 3.5 Mean concentrations of dissolved and particulate B-vitamins. Dissolved (blue) and particulate (red)
mean concentrations (pM) were plotted at each station. Samples were binned into three depth categories at each
station: Surface (3m), Mid (3m-DCM), and DCM. Some stations did not have a sample in one bin, but there were no
stations with two samples in one bin. The vertical gray line is at the Gibraltar Strait and the vertical blue line is at the
Sicily Strait.
65
Vitamin B
7
and the amino acid methionine show consistent partitioning between the
dissolved/particulate pools across the sampling transect (Figure 3.5). For example, 75-100% of
the total vitamin B
7
pool was found in the dissolved fraction while most (60-100%) of the amino
acid methionine was in the particulate pool. The substantial difference between the observed
concentrations ranges for dissolved and particulate B
7
(8-1970 pM and 1.7-27 pM respectively)
suggest that B
7
accumulates in the dissolved pool. This is potentially due to the lower incidence
of B
7
auxotrophy observed in phytoplankton, compared to vitamins B
1
and B
12
, reported from
both cultures and –omics based studies (Croft et al. 2006; Sañudo-Wilhelmy et al. 2014).
However, our results were obtained in a single cruise under spring conditions, therefore further
research will be required to establish the extent of the high dissolved B
7
concentrations observed
here. Particulate methionine concentrations ranged from 0.7-21 pM, and dissolved
concentrations ranged from B.D.L.-2.8 pM (Figure 3.5). Unlike B
7
, which is a coenzyme,
methionine is an essential amino acid across all domains of life (Berg et al. 2007). As methionine
is required for protein synthesis, its standing stock within the cell is subjected to a high turnover
rate and must be maintained at a sufficient intracellular concentration that allows this process to
proceed (Berg et al. 2007). Further, microbial uptake of amino acids, both as a nutrient/energy
source and for use in protein synthesis, is well documented in marine systems, which is likely
responsible for the low observed dissolved methionine standing stock (Keil and Kirchman 1991).
Unlike vitamin B
7
and methionine, tB
1
, HMP, and tB
12
show less constant partitioning
trends between the two pools, suggesting more active transfer between the two fractions. Areas
where most of the vitamins where found in the particulate pool were locations with high
phytoplankton biomass (DCM) while regions with high bacterial abundance had low particulate
B-vitamin concentrations (Figure 3.5). At the DCM, tpB1, HMP, B
7
, Met and tpB
12
were
66
significantly positively correlated with Chl-a concentration, providing evidence that
phytoplankton, drive the observed particulate concentrations, either through B-vitamin uptake or
synthesis, in regions where their activity is maximal (Sañudo-Wilhelmy et al. 2014). At the
surface of the Mediterranean-Atlantic Ocean transect, significant negative correlations exist
between heterotrophic bacterial abundance and particulate concentrations of tpB
12
, methionine,
and HMP (Pearson’s r = -0.83,-0.7, and-0.74 respectively; Figure 3.5) LNA cells compose the
majority of the total heterotrophic bacterial abundance at the locations where there is elevated
particulate B-vitamin concentrations (Figure 3.2), implying that LNA bacteria are responsible for
the highest particulate B-vitamin concentrations.
3.4.4 Particulate B-Vitamin Congener Relative Abundance
The particulate B-vitamin congener concentrations reported here are the first ever basin-
scale oceanographic measurements of the distributions of these compounds (Figure 3.5 and
Figure 3.6). The observed patterns of the relative abundance of the B-vitamin congeners to the
total particulate pool were substantially different than those observed in the dissolved pool
(Figure 3.4 and Figure 3.7). The distribution and relative contribution of the thiamin congeners
to the total particulate pool was remarkably constant across the entire transect despite the large
concentration fluctuations observed in the total particulate B
1
fraction (Figure 3.5 and Figure
3.7). In contrast to the dissolved fraction where vitamin B
1
was the most abundant chemical
form, within the particulate, B
1
and TMP are present in relatively equal abundances (IQR 47-
59% and 34-43% of tpB
1
respectively), and TPP was a minor contributor (IQR 4-12%) to the to
the total particulate pool. TPP only became the most dominant congener (49% of tpB
1
) at the
DCM at the Gibraltar strait (sta 24), which is also the location where the highest phosphorus
concentration was observed (Figure 3.2 and Figure 3.7). Intense phosphorus limitation has been
67
repeatedly demonstrated in the Mediterranean Sea (Krom et al. 1991; Krom et al. 2010;
Thingstad et al. 2005; Thingstad and Rassoulzadegan 1995). It is possible that the maximal
observed relative abundance of TPP is caused by an increase in metabolic activity and therefore
an increase in thiamin demand caused by the alleviation of phosphorus stress.
Figure 3.6 Particulate B-vitamin depth profiles. Particulate B-vitamin concentrations (pM) plotted as a function of
depth for all analytes at all stations. The color of the profiles indicate their regional location with profiles from the
eastern region in orange hues, profiles from the western region in blue hues, and profiles from the Atlantic Ocean in
green hues.
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Particulate Concentration (pM)
Depth (m)
Station
2
9
17
20
22
24
25
28
29
DCM
B
1
TMP TPP HMP B
7
MET AB
12
CB
12
HB
12
MB
12
68
Figure 3.7 Relative abundances of particulate thiamin and cobalamin congeners. Relative abundances were
calculated as percent of the total particulate thiamin and cobalamin pools respectively. Samples were binned into four
depth categories at each station: Surface (3m), Mid (3m-DCM), DCM, and Deep (DCM-300m). Some stations did not
have a sample in one bin, but there were no stations with two samples in one bin. The vertical gray line is at the Gibraltar
Strait and the vertical blue line is at the Sicily Strait.
The relative contribution of the different cobalamin congeners to the particulate pool is
also substantially different from the observed trends in the dissolved phase (Figures 2,4); HB
12
replaces MB
12
as the dominant particulate congener with an IQR of 62-86% of the tpB
12
fraction
(Figure 3.7). The other cobalamin congeners make up the minority of the tpB
12
fraction in all but
two locations with IQRs of 7-24% AB
12
, 3-14% MB
12
, and 0-1% CB
12
(Figure 3.7). AB
12
and
MB
12
become the most abundant chemical
29 28 25 24 22 20 17 9 2
0
25
50
75
100
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50
75
100
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50
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100
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50
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100
29 28 25 24 22 20 17 9 2
Station
Relative Abundance (%)
Particulate Congeners
Surface Mid DCM Deep
CB
12
HB
12
AB
12
MB
12
B
1
TMP
TPP
69
Station Depth(m) Latitude Longitude Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D.
2 3 34.073 31.413 97.0 52.2 67.3 29.9 13.9 5.82 51.7 47.4 9.71 6.49 8.41 7.10 4.15 3.11 0.779 19.2 9.56 2.42 1.12
2 25 34.073 31.413 45.8 11.5 27.1 5.32 10.3 8.05 9.25 1.93 3.77 1.10 0.710 0.819 0.0667 0.0524 0.463 0.025 2.16 1.09 0.351 0.171
9 3 37.715 19.772 62.2 21.3 34.4 5.84 7.84 10.3 35.0 5.65 2.86 0.0863 3.31 1.30 1.57 0.950 13.0 3.71 0.423 0.371
9 50 37.715 19.772 16.2 2.87 17.2 5.19 1.27 0.322 4.51 1.36 4.81 1.38 1.61 0.698 0.0961 0.00277 0.177 9.01 2.37 0.345 0.0585
9 95 37.715 19.772 138 5.82 48.7 4.00 24.9 8.96 16.8 5.46 4.76 2.08 1.56 0.699 0.0374 0.00259 2.29 0.57 0.0410
9 200 37.715 19.772 222 56.9 128 12.1 20.3 3.91 106 20.3 17.0 5.61 19.0 1.93 12.8 0.861 41.4 13.7 5.18 0.716
17 3 41.489 7.492 53.3 10.8 39.3 5.95 2.25 0.571 9.08 2.88 3.74 0.694 2.33 1.47 0.307 0.0535 5.64 0.758 1.55 0.284
17 60 41.489 7.492 205 36.9 157 35.1 16.8 10.7 84.8 9.05 27.1 1.49 21.3 10.0 6.86 0.712 0.0854 4.32 0.231 6.89 5.74
20 3 38.413 5.215 16.1 1.97 11.8 3.15 1.59 0.206 2.37 0.0889 2.89 0.469 2.25 0.357 1.32 0.269 0.0017 2.42 0.318 0.194 0.109
20 25 38.413 5.215 122 32.3 56.5 28.3 9.67 1.74 12.5 4.39 1.78 0.978 5.61 0.760 0.240 0.110 0.0387 0.0162 3.14 0.653 0.0276 0.00917
20 65 38.413 5.215 2.77 0.805 4.52 1.25 0.47 2.55 0.0108 3.25 0.466 4.48 0.479 0.112 0.0115 0.0298 0.0168 2.92 1.56 0.0625 0.0236
20 100 38.413 5.215 68.0 19.5 42.5 8.49 3.54 1.73 40.0 10.4 10.9 4.04 13.5 0.701 4.57 2.54 5.83 1.44 1.57 0.503
22 3 37.010 0.000 6.32 1.43 6.45 0.409 1.75 2.09 0.269 2.37 0.417 3.14 0.282 0.330 0.232 1.59 0.176 0.349 0.149
22 55 37.010 0.000 8.20 1.51 12.1 2.82 12.9 7.13 4.74 1.32 4.68 3.43 11.8 0.341 1.33 0.218 0.0198 5.10 1.42 0.655 0.056
24 3 35.986 354.633 51.9 6.42 31.0 3.41 24.0 23.6 13.9 3.67 5.13 1.06 3.43 1.09 0.184 0.246 2.16 0.586 0.0978 0.00197
24 12 35.986 354.633 42.3 15.1 35.9 2.53 3.58 3.27 42.5 11.51 11.5 3.71 11.8 4.99 6.35 1.79 0.374 8.23 1.67 1.79 0.450
24 25 35.986 354.633 7.80 2.45 7.75 3.44 15.1 6.00 1.87 0.669 1.10 0.356 0.094 0.016 1.97 0.728 0.0958 0.0306
25 3 36.017 352.033 47.5 1.53 25.5 2.87 1.85 1.17 8.39 0.749 5.67 1.20 0.887 0.0958 0.467 0.236 0.0693 1.81 0.297 0.346 0.158
25 50 36.017 352.033 39.9 1.65 31.8 8.71 7.00 1.13 33.4 3.57 6.77 0.453 12.0 6.45 1.77 0.154 4.38 0.600 2.38 0.931
25 75 36.017 352.033 14.6 5.56 17.8 10.3 4.40 2.95 41.1 10.9 7.70 5.44 15.5 3.20 2.11 0.989 0.0314 4.16 1.75 1.27 0.901
28 3 33.021 347.759 36.0 7.19 27.9 2.62 7.85 1.01 5.91 1.90 8.22 3.00 3.06 0.708 0.820 0.641 6.38 0.886 0.232 0.169
28 50 33.021 347.759 44.4 8.36 46.5 6.59 1.13 21.7 5.05 8.56 2.42 6.36 1.73 2.11 0.689 9.11 4.07 2.46 0.124
28 85 33.021 347.759 27.6 1.97 20.7 6.11 6.46 45.6 2.79 8.35 1.32 16.1 3.00 4.54 1.569 1.22 3.39 1.10 0.331 0.270
28 200 33.021 347.759 3.54 1.24 5.64 0.600 1.20 0.302 7.31 1.87 1.68 0.293 1.97 1.55 0.520 0.107 0.0115 0.00899 1.21 0.127 0.200 0.0228
29 3 31.250 347.023 52.2 2.50 34.2 7.43 2.55 44.4 4.23 14.1 2.59 8.03 1.76 3.03 0.231 0.146 0.0615 10.4 2.70 0.427 0.0769
29 50 31.250 347.023 63.1 16.1 47.2 11.5 1.97 8.06 1.47 6.34 1.62 2.71 1.58 1.51 0.988 6.95 1.00 1.80 0.896
29 70 31.250 347.023 80.2 19.6 47.2 2.70 5.91 0.110 10.5 1.42 2.43 0.542 1.39 0.682 0.411 0.488 0.0321 1.63 0.332 0.0244 0.0165
AB
12
CB
12
HB
12
MB
12
B
1 TMP TPP HMP
B
7 Methionine
Table 3.3 Particulate B-vitamin Concentrations (pM). Mean concentrations are provided for all analytes at all
stations. Blank cells indicate that the analyte was below detection limits at that location.
70
forms (48% of tpB
12
) at the DCM at one station in the Atlantic (sta. 28) and at the DCM (38% of
tpB
12
) at the North Balearic Front (sta. 17). Local chlorophyll maxima were observed at both of
these locations relative to their adjacent stations and high heterotrophic bacterial abundances
were also present. These observations potentially indicate that a distinct microbial assemblage
with higher microbial activity, and thus a higher demand for the active forms of cobalamin was
present at these locations. The preponderant abundance of the non catalytically active form of
cobalamin (HB
12
) found in most of the particulate samples analyzed in our cruise is likely driven
by the intracellular catalysis-regeneration cycle of cobalamin, where HB
12
is generated as a result
of the methyl transfer in B
12
-dependant methionine synthesis (Koutmos et al. 2009). However,
we cannot rule out that the enrichment of this chemical form of cobalamin in the particulate
fraction only reflects the uptake of HB
12
from the dissolved pool, as this vitamin form is the
second most abundant congener found in the water column (Figure 3.4).
3.4.5 B-Vitamin Quotas
B-vitamins are essential metabolic coenzymes, yet their cellular requirements in marine
phytoplankton and bacteria have not been directly measured; therefore, in order to gain an
understanding of these requirements, we calculated bulk community B-vitamin quotas from
regions with distinct microbial communities. Quotas were calculated using observed particulate
B-vitamin normalized to particulate organic nitrogen (PON) concentrations determined in the
photic zone (upper 200 m) at nine stations in the Mediterranean Sea (Figure 3.8 and Table 3.4).
PON was used, as its concentrations are known have less variability due to carbon fixation than
POC (Menden-Deuer and Lessard 2000; Smirnov and Revkova 2002). In order to make the best
possible approximation of cellular B-vitamin quotas, we chose to focus on regions where we
observed distinct microbial communities so that the microbial diversity represented by each
71
quota would be maximally constrained (Figure 3.2). The transect was separated into three
regions: The Eastern region, where Prochlorococcus and LNA were most abundant; the Western
region, where Chl-a and nutrients had maximal values and Synechococcus and heterotrophic
bacteria were more abundant; and the Atlantic Ocean region, that includes all stations west of the
Gibraltar Strait where the community was mostly composed of Prochlorococcus,
picoeukaryotes, LNA, and heterotrophic bacteria (Figure 3.1 and Figure 3.2). The normalized
intracellular B-vitamin quotas vary between one and two orders of magnitude within each region
(Figure 3.8), as can be expected from bulk environmental samples containing a diverse group of
organisms with diverse metabolic rates and requirements (Smirnov and Revkova 2002). The
maximal quotas were detected as statistical outliers in the Atlantic (for tpB
1
and B
7
), and in the
Western Region of the Mediterranean (for HMP, B
7
, methionine and tpB
12
; shown as black filled
-circles in Figure 3.8). While these points are statistical outliers, they have biological relevance
as they were observed at DCM depths. In the Eastern region, although not represented as
statistical outliers, the maximal quotas (except for B
7
and tpB
12
) were also measured at the DCM
(Figure 3.2 and Figure 3.8). The maximal B-vitamin quotas measured at DCMs range between 3
and 10 times higher than the median quota values obtained for the rest of the community, which
provides evidence that B-vitamin requirements might fluctuate based on rates of microbial
activity and/or species composition (Menden-Deuer and Lessard 2000; Smirnov and Revkova
2002). We hypothesized that the maximal quotas measured at DCM depths might represent the
“division quota” which is the intracellular B-vitamin concentration at which the cells in a
population are actively growing and dividing (Smirnov and Revkova 2002), while the vitamin
quotas measured at other depths could be considered “minimum quota” where biochemical and
physiological processes are maintained at a basal level (Smirnov and Revkova 2002). Inter-
72
regional trends within the medians of the B-vitamin quotas are immediately apparent, and are
likely due to each region’s distinct microbial community (Figure 3.2 and Figure 3.8). The
medians for all analytes (Figure S4) share a common trend where the Eastern Mediterranean and
the Atlantic Ocean have higher values than the Western region (Figure 3.8). Total thiamin (tpB
1
)
had the highest quota in the Eastern region, with a median quota value of 350pmole
vitamin/µmole PON, which was significantly greater than in the Western region (200 pmole
vitamin/µmole PON) (p-value= 0.042). The tpB
1
quotas were statistically similar in the Western
and Atlantic regions where the median quota value for the latter was 190pmole vitamin/µmole
PON (Figure 3.8). The thiamin precursor moiety HMP has median quotas that are five fold more
abundant in the Eastern region (55 pmole vitamin/µmole PON) and the Atlantic (49 pmole
vitamin/µmole PON) than in the western region (11 pmole vitamin/µmole PON). Despite the
differences in the medians, the IQR (Figure 3.8) of the Atlantic and Western region overlaps,
suggesting that the distribution of a group of organisms (such as heterotrophic bacteria which
have similar abundances in the Atlantic and the Western Region) with similar B-vitamin quotas
spans both of these regions (Figure 3.8). Similarly, the quotas of tpB
12
have
73
Figure 3.8 Particulate B-vitamin Quotas. Quotas were calculated using observed particulate B-vitamin
concentrations normalized to observed particulate organic nitrogen concentrations (pmole vitamin/µmole PON). Samples
were binned into three regions based on the biogeography of each. The Atlantic region (green) includes all stations west
of the Gibraltar Strait. The West Region (blue) includes all stations between the Gibraltar Strait and the Sicily Strait. The
East Region (orange) includes all stations East of the Sicily Strait. The horizontal line in each boxplot represents the
median, and the upper and lower bounds of each box indicate the interquartile range (IQR). The black circles indicate
statistical outliers.
0
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atl west east atl west east atl west east atl west east
atl west east atl west east atl west east atl west east
atl west east atl west east atl west east atl west east
Vitamin Quota (pM vitamin/uM PON)
B
1
TMP TPP tB
1
HMP MET B
7
AB
12
CB
12
HB
12
MB
12
tB
12
74
Station Depth(m) Latitude Longitude PON (µM) Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D.
2 3 34.073 31.413 0.399 243 131 169 74.8 34.8 14.6 129 119 24.3 16.3 21.1 17.8 10.4 7.79 1.95 48.1 23.9 6.05 2.80
2 25 34.073 31.413 0.485 94.6 23.8 55.9 11.0 21.2 16.6 19.1 3.98 7.78 2.28 1.46 1.69 0.138 0.108 0.956 0.0509 4.47 2.26 0.724 0.353
9 3 37.715 19.772 0.493 126 43.3 69.7 11.8 15.9 21.0 71.1 11.5 5.80 0.175 6.72 2.64 3.19 1.93 26.4 7.53 0.859 0.752
9 50 37.715 19.772 0.487 33.3 5.90 35.4 10.7 2.61 0.662 9.25 2.79 9.88 2.84 3.31 1.43 0.197 0.00569 0.363 18.5 4.87 0.707 0.120
9 95 37.715 19.772 0.422 328 13.8 115 9.48 59.1 21.2 39.8 12.9 11.3 4.94 3.69 1.66 0.0887 0.00615 5.42 1.35 0.0972
9 200 37.715 19.772 0.155 1440 368 831 78.3 131 25.3 684 131 110 36.3 123 12.5 83.0 5.57 268 88.5 33.5 4.63
17 3 41.489 7.492 0.819 65.1 13.2 48.0 7.27 2.75 0.698 11.1 3.51 4.57 0.847 2.84 1.80 0.374 0.0653 6.89 0.926 1.89 0.347
17 60 41.489 7.492 0.712 287 51.8 220 49.3 23.5 15.0 119 12.7 38.0 2.10 29.9 14.1 9.63 1.00 0.120 6.07 0.325 9.68 8.06
20 3 38.413 5.215 0.938 17.1 2.10 12.6 3.36 1.70 0.220 2.53 0.0949 3.08 0.500 2.40 0.380 1.41 0.287 0.00183 2.58 0.339 0.207 0.116
20 25 38.413 5.215 0.685 178 47.1 82.5 41.3 14.1 2.54 18.2 6.4 2.60 1.43 8.18 1.11 0.350 0.161 0.0564 0.0236 4.58 0.953 0.0403 0.0134
20 65 38.413 5.215 0.710 3.91 1.13 6.37 1.76 0.658 3.60 0.0152 4.58 0.656 6.31 0.674 0.158 0.0161 0.0419 0.0236 4.11 2.20 0.0880 0.0333
20 100 38.413 5.215 0.349 195 55.9 122 24.3 10.1 4.95 115 29.8 31.1 11.6 38.6 2.01 13.1 7.26 16.7 4.12 4.48 1.44
22 3 37.010 0.000 0.937 6.74 1.53 6.88 0.437 1.87 2.23 0.287 2.53 0.445 3.36 0.301 0.352 0.248 1.70 0.188 0.373 0.159
22 55 37.010 0.000 1.23 6.69 1.23 9.87 2.30 10.5 5.82 3.86 1.08 3.82 2.80 9.64 0.278 1.08 0.178 0.0162 4.16 1.16 0.534 0.046
24 3 35.986 354.633 0.536 96.8 12.0 57.9 6.35 44.7 44.1 26.0 6.84 9.57 1.98 6.40 2.03 0.343 0.459 4.03 1.09 0.182 0.004
24 12 35.986 354.633 0.332 127 45.5 108 7.62 10.8 9.85 128 34.7 34.7 11.2 35.6 15.0 19.1 5.41 1.13 24.8 5.04 5.38 1.36
24 25 35.986 354.633 0.201 38.8 12.2 38.6 17.1 75.2 29.9 9.30 3.33 5.50 1.77 0.467 0.0793 9.83 3.63 0.477 0.152
25 3 36.017 352.033 0.363 131 4.22 70.3 7.91 5.08 3.23 23.1 2.06 15.6 3.32 2.44 0.264 1.29 0.651 0.191 4.97 0.818 0.954 0.434
25 50 36.017 352.033 0.354 113 4.66 89.8 24.6 19.8 3.19 94.3 10.1 19.1 1.28 34.0 18.2 4.99 0.435 12.4 1.70 6.72 2.63
25 75 36.017 352.033 0.472 31.0 11.8 37.8 21.8 9.32 6.25 87.0 23.1 16.3 11.5 32.9 6.78 4.48 2.10 0.0665 8.82 3.72 2.69 1.91
28 3 33.021 347.759 0.501 71.8 14.3 55.6 5.23 15.7 2.02 11.8 3.78 16.4 5.99 6.10 1.41 1.64 1.28 12.7 1.77 0.464 0.337
28 50 33.021 347.759 0.391 113 21.4 119 16.8 2.89 55.5 12.9 21.9 6.18 16.2 4.43 5.40 1.76 23.3 10.4 6.29 0.317
28 85 33.021 347.759 0.357 77.2 5.52 57.9 17.1 18.1 128 7.80 23.4 3.70 45.1 8.39 12.7 4.39 3.40 9.50 3.09 0.927 0.757
28 200 33.021 347.759 0.176 20.1 7.07 32.1 3.41 6.80 1.71 41.6 10.6 9.57 1.67 11.2 8.81 2.96 0.606 0.0653 0.0511 6.88 0.720 1.13 0.130
29 3 31.250 347.023 0.381 137 6.56 89.8 19.5 6.69 117 11.1 37.0 6.80 21.1 4.61 7.94 0.606 0.384 0.161 27.3 7.08 1.12 0.202
29 50 31.250 347.023 0.352 179 45.7 134 32.6 5.59 22.9 4.18 18.0 4.60 7.69 4.48 4.28 2.80 19.7 2.85 5.11 2.54
29 70 31.250 347.023 0.302 265 64.8 156 8.93 19.6 0.362 34.6 4.69 8.03 1.79 4.58 2.25 1.36 1.61 0.106 5.38 1.10 0.0807 0.0547
AB
12
CB
12
HB
12
MB
12
B
1 TMP TPP HMP
B
7 Methionine
Table 3.4 B-vitamin Quotas. Mean particulate concentrations (pM) were normalized with observed particulate
organic nitrogen concentrations (included in table, µM). B-vitamin quota units are pmole vitamin/µmole PON.
Blank cells indicate that the analyte was below detection limits at that location.
75
overlapping IQRs in Western and Atlantic regions, but the median quota of the Western region is
five times lower than the Atlantic (45.7 and 9.4 pmole vitamin/µmole PON respectively); the
observed median values for the Eastern region is eight fold higher than the western region (71.5
pmole vitamin/µmole PON). The B
7
quotas are roughly two and four fold higher in the Eastern
region and Atlantic Ocean than in the western Region (10.6 East, 17.2 Atlantic, 4.6 West pmole
vitamin/µmole PON), but the IQRs of all regions overlap suggesting that there are not significant
differences among the regions (Figure 3.8). The IQRs of methionine for the three regions also
overlap, but the median value in the Atlantic Ocean (13.7 pmole vitamin/µmole PON) was two
fold greater than that in the Western (6.4 pmole vitamin/µmole PON) and three fold greater than
in the Eastern Mediterranean (5.2 pmole vitamin/µmole PON).
Our data indicate that median B-vitamin quotas are highest in areas where oligotrophic,
open ocean microbial communities are present. Prochlorococcus and LNA are the numerically
dominant microbes in the Eastern Region of the Mediterranean Sea where the median quotas for
all measured analytes were highest, and, to a lesser extent, in the Atlantic Ocean where median
quota values were always higher than the Western region (Figure 3.2, Figure 3.8,S4). These
regions are oceanographically similar, and multiple comparisons have been made between the
Eastern Mediterranean and the oligotrophic Atlantic Ocean (Krom et al. 2010; Siokou-Frangou
et al. 2010; Tanaka et al. 2011; Tanaka et al. 2007; Thingstad et al. 2005). We hypothesize that
the elevated quotas measured in these regions could represent the vitamin requirements and de
novo synthesis of SAR11, SAR86, and Prochlorococcus. SAR11 is known to require HMP, B
7
,
and methionine as a reduced sulfur source (Carini et al. 2014; Giovannoni et al. 2005); SAR86 is
known to require B
1
, B
7
, methionine (protein synthesis), and B12 (Dupont et al. 2012); and
76
Prochlorococcus is known to be able to synthesize all the vitamins it requires, including the
coenzymes and the amino acid measured here (Zinser et al. 2009). These organisms have been
subjected to substantial genome streamlining, which has forced them to rely on other members of
the community to obtain necessary metabolites (Dupont et al. 2012; Evans et al. 2015;
Giovannoni et al. 2005; Morris et al. 2012). Additionally, B-vitamin mediated community
interactions can be inferred from transcriptomics based approaches (Gomez-Consarnau et al.
2017). It is likely that the transfer of B-vitamins between producers and consumers is especially
tightly coupled in oligotrophic regions (such as the Eastern Basin) where dissolved vitamin
concentrations can be order of magnitude lower than the maximum observed values (observed in
the western region; Figure 3.2).
In the Western region of the Mediterranean, we measured the highest levels of
phytoplankton biomass (as Chl-a) and heterotrophic bacteria, and the most abundant
cyanobacteria group was Synechococcus (Figure 3.2). However, median B-vitamin quotas
measured in that region were some of the lowest (Figure 3.8). The abundant heterotrophic
bacteria in this region likely represent both a source and a sink for B-vitamins, however vitamin
requirements and synthesis capacity is paraphyletic, and variable for each vitamin, so it is not
possible to determine the exact abundances of B-vitamin producers and auxotrophs within the
diverse heterotrophic bacteria (Sañudo-Wilhelmy et al. 2014). However both producers and
auxotrophs are known to be found within groups including Flavobacteria, Rhodobacerales,
SAR116, and Bacteriodetes (Gomez-Consarnau et al. 2017; Sañudo-Wilhelmy et al. 2014),
which have been observed to be abundant in this region of the Mediterranean (Pinhassi et al.
2006; Vila-Costa et al. 2012). Additionally, strong transcriptomic evidence for B-vitamins
mediated interactions between groups of heterotrophic bacteria has recently been demonstrated
77
(Gomez-Consarnau et al. 2017). The bacterial activity in this region was the highest observed in
the entire transect (Figure 3.9). Taken together the high rate of bacterial activity and complex
microbial community including both B-vitamin synthesizers and auxotrophs, potentially results
high biomass turnover leading to low quotas and the maximal observed concentrations of
dissolved B-vitamins found in this region.
Figure 3.9 Distribution of Bacterial Production. Bacterial Production (gC/(L*Day)) measured by the leucine
incorporation is plotted as a function of depth and longitude. The vertical gray line is at the Gibraltar Strait and the
vertical blue line is at the Sicily Strait.
Directly measured B-vitamin quotas have yet to be determined for individual species of
marine microbes. A literature survey of existing B-vitamin quotas from phytoplankton estimated
from culture based B-vitamin uptake experiments in HAB species (Gobler et al. 2007; Tang et al.
2010) and Prymnesiophytes (Paerl et al. 2015; Paerl et al. 2016), in addition to directly measured
environmental quotas from a coastal region using a bioassay (Carlucci and Bowes 1972)
determined that the mean reported POC normalized B-vitamin quotas (pmole vitamin/µmole
POC) are 0.65±0.79 B
1
, 0.08±0.14 B
7
, and 0.02±0.05 B
12
. The mean observed bulk POC
normalized vitamin quotas (also in pmole vitamin/µmole POC) measured in the natural
communities sampled during the Mediterranean-Atlantic transect are 31±28 tpB
1
, 2.5±3.4 B
7
,
Leucine Incorporation gC/(L*day)
Picoeukaryote Abundance (cells/ml)
FIGURE 2.S5
78
and 3.0±3.5 tpB
12
. The two order of magnitude discrepancy between mean uptake/bioassay
based quotas and the directly measured quotas may indicate that the B-vitamin requirements in
marine microbes could have been substantially underestimated using these techniques. However,
there is innate variability in the concept of a cellular quota. It has been demonstrated with
cellular nutrient quotas that fluctuations of the magnitude that we observed are not uncommon
depending on changes in metabolic demand caused by exogenous nutrient availability, growth
rates, or cellular division (Smirnov and Revkova 2002). The dose response methods used to
calculate uptake rates and cellular vitamin quotas do not take into account these changes in
metabolic potential, therefore these values can be considered basal, minimal quotas (Smirnov
and Revkova 2002). In contrast to the tightly controlled laboratory monocultures (Carini et al.
2014; Gobler et al. 2007; Paerl et al. 2015; Paerl et al. 2016; Tang et al. 2010), our directly
measured bulk environmental quotas include organisms from all three domains of life with
differing B-vitamin requirements (and synthesis capabilities) caused by diverse metabolic modes
(heterotrophy vs phototrophy) and variable growth rates found in the microbial assemblage.
Future research will need to include both uptake based methods and directly measured quotas to
gain a more complete understanding of cellular B-vitamin requirements.
3.4.6 Dissolved B-Vitamin Based Linear Regression Modeling
It has been repeatedly demonstrated in both field and laboratory experiments that
amendments of B-vitamins can cause shifts in planktonic assemblages, increases in growth rates,
and increases in biomass production, suggesting that B-vitamins can be limiting growth factors
for both marine prokaryotes and eukaryotes (Bertrand and Allen 2012; Bertrand et al. 2012a;
Bertrand et al. 2012b; Bertrand et al. 2015; Bertrand et al. 2007; Gobler et al. 2007; Koch et al.
2013; Paerl et al. 2015; Paerl et al. 2016; Panzeca et al. 2006). However, it has yet to be shown
79
that the availability of dissolved B-vitamin congeners, and some of their synthetic precursors,
have the ability to influence the environmental distribution of microbial species. In order to
establish this link, we used linear regression modeling to establish a correlative link between the
abundances of known B-vitamin auxotrophs (e.g., SAR11, SAR86, and Picoeukaryotes) and with
B-vitamin producers (e.g., Prochlorococcus and Synechococcus) found along the Mediterranean-
Atlantic Ocean transect with the concentrations of the dissolved B-vitamins and their congeners.
We also included in the analysis other known biomass controlling parameters such as inorganic
nutrients (nitrate and phosphate) concentrations and light levels (as photosynthetic active
radiation (PAR)). We applied a stepwise linear regression algorithm to exhaustively calculate
models using all dissolved B-vitamin congeners (B
1
, TMP, TPP, B
7
, AB
12
, MB
12
, HB
12
, CB
12
),
total dissolved thiamin and cobalamin (tdB
1
and tdB
12
), HMP (B
1
synthesis precursor), the amino
acid methionine, dissolved nutrients (NO
3
, PO
4
), and PAR as variables to correlate with the
abundance of LNA cells, heterotrophic bacteria, Picoeukaryotes, Prochlorococcus, and
Synechococcus (R Core Team 2015). Models were applied across the entire Mediterranean-
Atlantic Ocean transect, and on a regional basis (Figure 3.1 and Figure 3.2) to capture areas
where specific organisms were observed to be the most abundant. All of the vitamin congeners,
total thiamin and cobalamin were also included as potential variables for each model; the
selection of total thiamin or cobalamin indicates that there was only a significant correlation with
the summed total pool, not with the individual congeners. The only statistically significant
models that were produced using this correlative approach are presented here. Those models
were produced for LNA and Prochlorococcus in the Eastern Mediterranean and the Atlantic
Ocean, Picoeukaryotes in the Atlantic Ocean, heterotrophic bacteria in all regions, and
Synechococcus in the Western region of the Mediterranean (Figure 3.10). Our results indicate,
80
for the first time, strong correlations between dissolved B-vitamin congeners and the
geographical distribution of different microbial populations in the area of study (Figure 3.10).
Figure 3.10 Linear regression modeling using dissolved B-vitamin concentrations and inorganic nutrients (N, P,
PAR) predicts microbial abundances. All x-axis are observed abundances, and all y-axis are modeled abundances. Black
points represent models including dissolved B-vitamins. Red points represent models including only the inorganic
nutrients found in each model. Adjusted R squared values, and the model equation are provided on each plot for both
models. All values have been normalized using the natural log. The blue line is the 1:1 line, points falling on this line have
been modeled accurately.
0
3
6
9
0.0 2.5 5.0 7.5 10.0
observed
modeled
Syn West Region
0
4
8
0 3 6 9 12
observed
modeled
Pro East Region
3
6
9
12
0 3 6 9 12
observed
modeled
Pro Atlantic Ocean
11.0
11.5
12.0
12.5
13.0
11.5 12.0 12.5
observed abundance
modeled abundance
LNA East Region
12.0
12.5
13.0
13.5
12.0 12.5 13.0 13.5
observed abundance
modeled abundance
LNA Atlantic Ocean
2.5
5.0
7.5
0.0 2.5 5.0 7.5 10.0
observed
modeled
Pico Euks Atlantic Ocean
aR
2
= 0.88
aR
2
= 0.38
aR
2
= 0.58
aR
2
= 0.39
aR
2
= 0.86 aR
2
= 0.74
aR
2
= 0.41
aR
2
= 0.84
aR
2
= 0.66
aR
2
= 0.93
aR
2
= 0.73
12.37+0.07tB
1
-0.7B
7
-0.49MET-1.38NO
3
+9.34PO
4
12.87-1.82NO
3
+7.31PO
4
13.65+0.29TMP-0.18TPP+0.47HMP
-0.31B
7
-5.32CB
12
-1.98HB
12
-1.81MET-3.87PO
4
13.06-3.58PO
4
10.8-44.49PO
4
8.14-0.32B
1
+0.29B
7
+8.81CB
12
-1.93HB
12
-5.68MET+2.31NO
3
-27.6PO
4
2.57+7.16NO
3
-61.09PO
4
+1.28PAR
5.41+1.04tB
1
+0.54B
7
-2.27HB
12
-4.45MET-22.3PO
4
10.24-23.7PO
4
18.24-3.69TPP+6.19HMP
-20.85NO
3
-89.28PO
4
-3.17PAR
13.29-15.37NO
3
74.35PO
4
-1.59PAR
12.0
12.5
13.0
13.5
12.5 13.0 13.5
observed
modeled
BAC West Region
12.5
13.0
13.5
14.0
12.5 13.0 13.5 14.0
observed
modeled
BAC Atlantic Ocean
aR
2
= 0.91
aR
2
= 0.31
14.37-0.10TMP+0.38HMP-0.25B
7
+4.91CB
12
-1.39HB
12
-1.61MET-2.39PO
4
13.57-2.95PO
4
aR
2
= 0.91
aR
2
= 0.81
13.69+0.56AB
12
-0.34HB
12
-6.01PO
4
-0.05PAR
13.91-6.58PO
4
-0.07PAR
12.0
12.4
12.8
12.0 12.4 12.8
observed
modeled
BAC East Region
aR
2
= 0.99
aR
2
= 0.79
12.88-0.20tB
1
+0.28B
1
-0.02B
7
-0.18CB
12
-0.70MET
-1.3NO
3
+7.69PO
4
-0.08PAR
13.17-1.33NO
3
+4.31PO
4
-0.11PAR
81
LNA cells were observed to be abundant in the Eastern region of the Mediterranean and in
the Atlantic Ocean, and it is known that the major components of this group, SAR11 and SAR86,
obligately require many compounds including B
1
, HMP (SAR11), B
7
, B
12
(SAR86), and the
amino acid methionine (Figure 3.2) (Carini et al. 2014; Carini 2013; Dupont et al. 2012). Within
the Eastern region where LNA was the predominant bacterial group present (~75% of the total
heterotrophic bacterial abundance) the strongest significant correlative model explained 93% of
the observed variability in their abundance using tdB
1
, B
7
, MET, nitrate, and phosphate
concentrations (Figure 3.10, p-value<0.001). In the same region, nitrate and phosphate alone
were only able to explain 73% of the observed variability in LNA abundance (Figure 3.10, p-
value<0.001), although that high correlation coefficient is heavily weighted by about 40% of the
data points (Figure 3.10). When the model was applied to total heterotrophic bacterial abundance
(including LNA) the results only differed slightly from the LNA model, predicting 99% of the
variability with B
1
, CB
12
, and PAR in addition to the variables included in the LNA model
(Figure 3.10). The inclusion of PAR in this model potentially indicates rhodopsin based
photoheterotrophy is active in this region, as has recently been described (Gomez-Consarnau et
al. 2017). In the Atlantic region LNA made up the majority of the heterotrophic bacterial
community with abundances ranging from 50-80% of total heterotrophic bacterial abundance.
Within this region 88% of the variability in observed LNA abundance could be modeled with
dissolved concentrations of TMP, TPP, HMP, B
7
, CB
12
, HB
12
, MET, and phosphate (Figure 3.10,
p-value<0.001). In contrast phosphate alone only weakly explained the variability in LNA
abundance (38%) (Figure 3.10, p-value<0.01). When total heterotrophic bacteria were modeled,
91% of the variability was explained with a model that used all the same variables as the LNA
model excluding TPP (Figure 3.10). Both models identified thiamin congeners as dependent
82
variables which is consistent with SAR86’s requirements, however SAR11 only requires HMP
(Carini et al. 2014). HMP is known to be produced as a degradation product of thiamin (Begley
1996; Begley et al. 1999; Edwards et al. 2017; Kraft and Angert 2017), and dissolved HMP
concentrations along the entire transect were significantly and positively correlated with tdB
1
,
TMP, and TPP (Pearson’s R = 0.76, 0.8, and 0.61 respectively). Therefore the observed
relationship with the thiamin congeners and total thiamin is likely indicative of a potential source
of HMP.
Picoeukaryotes were found to be most abundant in the Atlantic Ocean during our cruise,
with abundances reaching 1.3x10
4
cells/ml (Figure 3.11). Picoeukaryotic phytoplankton are a
size-based group (<3 microns), which include multiple phylogenetic groups; research has shown
that Prymnesiophytes are the dominant class in the region traversed by our Mediterranean-
Atlantic Ocean transect (Kirkham et al. 2011). Prymnesiophytes have been shown to be thiamin
auxotrophs, including strains that utilize different thiamin compounds including HMP, B
1
and/or
TPP in order to meet their thiamin requirements (Paerl et al. 2016). Additionally genomic
studies have shown that this class is known to require exogenous B
12
, but it has the ability to
synthesize B
7
(Sañudo-Wilhelmy et al. 2014). Within the Atlantic region, dissolved
concentrations of B
1
, B
7
, CB
12
, HB
12
, MET, nitrate, and phosphate explained 84% of the
variability in observed picoeukaryote abundance, compared to 66% when only nitrate,
phosphate, and PAR were used in the model (Figure 3.10). It is interesting that despite their
known ability to synthesize B
7
, the statistical model identified the availability of this vitamin as
an important biomass-dependent parameter, which suggests that an unidentified picoeukaryote
B
7
auxotroph might be present. Additionally the highest B
7
quota was measured in the microbial
83
community found in the Atlantic Ocean, highlighting the importance of this vitamin in that
marine region (Figure 3.2).
Figure 3.11 Distributions of Picoeukaryotes plotted as a function of depth and longitude. The vertical gray line is at
the Gibraltar Strait and the vertical blue line is at the Sicily Strait.
Prochlorococcus is a known B-vitamin synthesizer that was observed to be the most
abundant cyanobacteria in the Eastern region of the Mediterranean and the Atlantic Ocean during
our cruise (Figure 3.2; Sanudo et al 2014). We determined that its abundance is significantly
correlated with the concentrations of the dissolved B-vitamins it is known to synthesize (Moore
et al. 2007). This finding is not unusual; it has been shown that prokaryotes (including
Prochlorococcus) with B-vitamin synthesis capacity will preferentially assimilate/salvage and
ultimately utilize exogenous B-vitamins (instead of synthesizing them de novo) as an method to
reduce cellular energetic and nutrient demands (Bonnet et al. 2010; Evans et al. 2015; Koch et al.
2012). In the eastern region where Prochlorococcus abundance was about 40% of the
heterotrophic bacterial abundance (Prochlorococcus is not included in heterotrophic bacterial
abundance. e.g., Pro=0.4(Hetbac) ), 58% of the variability in Prochlorococcus abundance was
explained by the availability of dissolved concentrations of different forms of thiamin (TPP and
HMP), nitrate, phosphate, and PAR (Figure 3.10, p-value=0.013). Both TPP and HMP had
Leucine Incorporation gC/(L*day)
Picoeukaryote Abundance (cells/ml)
FIGURE 2.S5
84
significantly higher intracellular quotas in the Eastern region (p-values=0.048 and 0.003
respectively) than in the western region (Table 3.2), adding evidence to the correlative
relationship. In the Atlantic Ocean where Prochlorococcus abundance was about 15% of
heterotrophic bacterial abundance, 74% of the variability in Prochlorococcus abundance
depended on a more diverse chemical pool of B-vitamin congeners such as tdB
1
, B
7
, HB
12
, MET,
and only phosphate as an inorganic nutrient (Figure 3.10, p-value=0.001). When solely
inorganic nutrients were included in the model, the correlative power of analysis dropped to 39%
in the Eastern region of the Mediterranean and 41% in the Atlantic Ocean (Figure 3.10, p-
value=0.02 and 0.004). The presence of a correlation with dissolved HB
12
is unexpected as it has
been shown that most cyanobacteria synthesize and require pseudocobalamin, not cobalamin
(Heal et al. 2016; Helliwell et al. 2016). We did not measure pseudocobalamin in this study,
however based on experimental evidence that Prochlorococcus will utilize available exogenous
B-vitamins (Evans et al. 2015), and the known ability of prokaryotes to remodel corrinoids (Gray
and Escalante-Semerena 2009; Yi et al. 2012), it is possible that HB
12
is being assimilated and
then remodeled to pseudocobalamin. While it has been established that Prochlorococcus can
assimilate dissolved B-vitamins (Evans et al. 2015; Koch et al. 2012), it potentially more likely
that these observed correlations are due to release of particulate B-vitamins synthesized
Prochlorococcus into the dissolved pool, implying that Prochlorococcus is a source of B-
vitamins to the community.
In the Western region of the transect a diverse community of copiotrophic heterotrophic
bacteria and the cyanobacterium Synechococcus were present (Figure 3.2). Synechococcus is
known to have B-vitamin synthetic capability (Palenik et al. 2006), but was not found to have
any correlative relationships with any of the different B-vitamin congeners measured in the
85
dissolved fraction along the Mediterranean-Atlantic cruise. The model results showed that
phosphate concentration alone could account for 86% of its abundance (Figure 3.10, p-
value<0.001). While Synechococcus was the most numerically abundant cyanobacteria in the
Western region of the Mediterranean, it only comprises at most 4% of the heterotrophic bacterial
abundance (Figure 3.2). In contrast, the diverse heterotrophic bacterial community present in the
Western region is known to consist of both B-vitamin producers and auxotrophs (Pinhassi et al.
2006; Sañudo-Wilhelmy et al. 2014; Vila-Costa et al. 2012). We applied our model to
heterotrophic bacteria in this region to test if dissolved B-vitamin availability can be correlated
with the abundance of a diverse microbial community with each species having unique B-
vitamin requirements. The resulting model was able to explain 91% of the variability in
heterotrophic bacterial abundance using AB
12
, HB
12
, phosphate, and PAR, while phosphate and
PAR alone were able to account for 81% of the variability (Figure 3.10). The difference in
correlative power between model including B-vitamins and the model only including inorganic
factors is the smallest that we observed (10%), which suggests that the ability to use B-vitamins
as biomass predictors in a diverse community of heterotrophic bacteria with a complex
assemblage of B-vitamin requirements is reduced. However, the exclusive presence of cobalamin
species in the model is consistent with our understanding that cobalamin auxotrophy is the most
abundant form of B-vitamin auxotrophy found in marine microbial communities (Croft et al.
2006; Sañudo-Wilhelmy et al. 2014)
These correlative results highlight the importance of measuring the multiple chemical
forms of the B-vitamins, as no one congener or vitamin is able to explain the distributions of
different groups of organisms in the different oceanographic regions along the Mediterranean-
Atlantic Ocean transect. The inclusion of the thiamin congeners in all but one (Heterotrophic
86
Bactria, western region) of the models that included B-vitamins highlights this point, and
suggests that there are diverse requirements for the different thiamin congeners and that each
congener is not equally bioavailable to all organisms (Paerl et al. 2015). In contrast, dissolved
methionine appears to be widely utilized, as it was included in 70% of the models that also
included B-vitamins, and no significant regional differentiation in the methionine quotas were
observed (Figure 3.8). Concentrations of dissolved methionine are only rarely measured in the
ocean (Sañudo-Wilhelmy et al. 2012), yet it has been shown that this amino acid has special
importance to marine microbes as a reduced sulfur source in addition to an important protein
precursor (Carini et al. 2014). Additionally, methionine is the base component of S-adenosyl
methionine (SAM), which is an important methylation agent (Berg et al. 2007). Our results
suggest that more attention should be given to the compound in seawater, as it appears to be
highly important to microbial metabolism. Although MB
12
is the most abundant cobalamin
congener found in the dissolved fraction during our cruise (Figure 3.4), the two inactive forms of
vitamin B
12
(HB
12
and CB
12
) were the primary significant contributors to the biomass models
(Figure 3.4 and Figure 3.10). While HB
12
and CB
12
are catalytically inactive they are both
bioavailable, as HB
12
is the most abundant cobalamin congener found within the particulate
partition, and CB
12
is the congener that is most commonly added to microbial growth media
(Figure 3.4). Both of these congeners are minor contributors to the dissolved tdB
12
pool (IQRs
HB
12
11-19%, CB
12
3-7%), potentially because these forms are appear to be more labile than the
bioactive forms. The least abundant B
12
congeners appear to have the largest impact on the
community, which provides strong evidence that measuring the all B-vitamin congeners is very
important, since the biological response not only depends on actual concentration but in the
chemical form of the vitamins. Taken together, these findings indicate that B-vitamins and their
87
congeners likely have the ability to control the composition of the observed marine microbial
assemblage. If this is the case, it has substantial impactions for global biogeochemistry, as many
large phytoplankton that strongly contribute to carbon flux have been shown to be B-vitamin
auxotrophs (Croft et al. 2006; Sañudo-Wilhelmy et al. 2014). Therefore the ambient dissolved B-
vitamin concentrations, although six orders of magnitude lower compared to the concentrations
of macro nutrients, may be the key in determining the rates of carbon removal from the surface
ocean. In fact the availability of dissolved B-vitamins might be the key to the Paradox of the
Plankton (Hutchinson 1961).
Conclusions 3.5
The results of this study represent the most complete oceanographic B-vitamin dataset
assembled to date, including paired particulate and dissolved measurements of multiple
biochemical B-vitamin congeners. We use these data to establish spatial distributions of these
essential coenzymes spanning distinct microbiological and oceanographic regimes in the
Mediterranean Sea and the Atlantic Ocean. All B-vitamin congeners were present in both the
particulate and the dissolved pools across the transect, however, as has previously been reported,
large vitamin depleted regions were observed. Dissolved B-vitamins co-occurred in
geographically distinct patches where the concentrations were an order of magnitude higher than
the areas surrounded the geographical region. The dissolved B-vitamin patches occurred in
distinct water masses characterized by increases in biological productivity. Additionally, these
vitamin-enriched areas were generally found in zones with slightly negative N* values,
suggesting that phosphorus limitation was alleviated. The relative abundances of B-vitamin
congeners indicated a decoupling between the biochemical intracellular pool and the exogenous
dissolved fraction as MB
12
was the most abundant cobalamin congener in the dissolved pool
88
(IQR 52-70%) while HB
12
was most abundant in the intracellular fraction (IQR 62-86%). B
1
was
the most abundant congener in both the dissolved and particulate pools (IQR 71-83% dissolved;
47-59% particulate), however the magnitude of vitamin B
1
’s abundance was about 1.5 times
greater in the dissolved pool than in the particulate. Additionally, particulate B-vitamin quotas of
the bulk microbial community were calculated, which provide the first directly measured
estimates of environmental B-vitamin biological requirements in marine systems. Maximum
quotas were measured at DCM depths where the values were 2-10 times higher than the median
quota value observed along the whole transect, suggesting that increased rates of biological
activity increase B-vitamin requirements. Biogeographic trends in median quota values were also
observed. For example, the SAR11 clade of bacteria is known to require HMP, and the median
HMP quotas were between 4-7x higher in the regions with the greatest LNA (SAR11 and SAR86
clades) abundances, suggesting that the differentiation in B-vitamin quota values is caused by
differential microbial B-vitamin requirements. Finally, we used linear regression models to link
the abundances of microbes with B-vitamin auxotrophy to the exogenous availability of the
compounds that they require. We found that although the non-active forms (CB
12
and HB
12
)
were the least abundant dissolved congeners, they were the congeners most commonly correlated
with microbial abundance, suggesting that they are the most labile forms of cobalamin. Vitamin
B
7
was correlated in all but one of the significant models (heterotrophic bacteria in the western
region of the Mediterranean), which suggest that B
7
might be a more important player in
microbial growth than previously thought. Methionine was correlated in 70% of the significant
models, which is consistent with the known uptake of amino acids from the dissolved pool,
however the oceanic distribution of this amino acid is highly understudied and warrants more
attention.
89
These data represent a substantial advancement in our understanding of B-vitamin related
ecophysiology and biogeochemistry. However, the understanding of the biogeochemical and
ecological implications of B-vitamins in the ocean has been, and continues to be, hindered by the
limited information that exists. This study provides, for the first time, environmental paired
measurements of the standing stocks of the two pools of B-vitamins. Unfortunately, the rates of
the processes connecting these two pools (uptake, excretion, lysis, degradation, etc.) have only
been sparsely studied and are largely unknown. It is therefore likely that the relative size and
distribution of the particulate and dissolved pools of B-vitamins are not constant, and fluctuate
rapidly based on the microbial community present, and its specific rates of biochemical activity.
Future studies will be necessary to elucidate these rates.
Acknowledgements 3.6
We would like to thank Danielle Monteverde, Naomi Levine, Babak Hassanzadeh, Paige
Connell and Yubin Raut, for their help and advice with sample analysis, statistical analysis, and
manuscript preparation. Additionally we thank the captain and crew of the R/V Sarmiento de
Gamboa for their assistance in sample collection. Financial support for this research was
provided by NSF Biological Oceanography grant number OCE 1435666.
90
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98
Chapter 4 B-vitamin intracellular quotas influence
marine microbial bloom succession.
By Christopher P. Suffridge
Coauthors: Laura Gómez Consarnau, Feixue Fu, Pingping Qu, David Hutchins, and
Sergio A. Sañudo-Wilhelmy
99
Abstract 4.1
The availability of B-vitamins directly influences the dynamics of the marine
microbial community. A vitamins addition experiment was conducted with water
collected at a long-term study site in the San Pedro Basin (SPOT) off the coast of Los
Angeles, CA. Experimental amendments were conducted under unusually nutrient
depleted conditions due to the 2015 El Niño event. Vitamins B
1
, B
7
, and B
12
were added
in addition to macronutrients at concentration ranges typical observed at SPOT under
upwelling conditions. Intracellular and dissolved B-vitamin analyses were conducted to
determine shifts in cellular B-vitamin requirements as a function of growth rate.
Treatments with B
12
and macronutrients had the greatest growth rates. We observed
vitamin B
12
co-limitation of the phytoplankton community, which caused two
pronounced community shifts. The initial dinoflagellete dominated community was
replaced by a bloom of the diatom Chaetoceros, which is a vitamin B
12
auxotroph. By the
final sampling at stationary phase the community had shifted to a Pseudonitzschia bloom
(B
1
, B
7
, and B
12
auxotroph), likely driven by secondary bacterial B-vitamin synthesis. B-
vitamin quotas from the different experimental treatments suggest differential B-vitamin
acquisition strategies where thiamin and biotin were predominantly synthesized de-novo,
while cobalamin was assimilated from the dissolved pool. We observed strong inter-
domain differentiation among the cobalamin congener relative abundance between the
fractions containing bacterioplankton (picoplankton fraction) and eukaryotic
phytoplankton (nanoplankton fraction). The relative abundances of the active forms of
cobalamin (AB
12
and MB
12
) were higher in the nanoplankton fraction, despite that the
total cobalamin concentrations were an order of magnitude lower than the picoplankton
fraction, suggesting that the lack of the cobalamin synthesis pathway in eukaryotes has
100
lead to lower cellular requirements for this growth factor. These data provide evidence
that growth rate substantially influences B-vitamin quotas and requirements in marine
microbes. Additionally, our results demonstrate that exogenous B-vitamin availability
strongly influences not only the growth rate, but also the species composition of the
microbial community, which has the potential to influence the strength of the biological
carbon pump.
Introduction 4.2
Phytoplankton and bacterioplankton are key players in oceanic ecology and
biogeochemical cycling, however the factors that determine planktonic species
succession are still not fully understood (Du and Peterson 2001). External factors
affecting phytoplankton dynamics have been studied extensively over the past 30 years
with work primarily focused on the importance of inorganic macro nutrients (e.g.,
N,P,Si), and trace metals (e.g., Fe) on algal and post-algal bloom dynamics (Boyd et al.
2000; Dugdale and Goering 1967; Falkowski et al. 1998; Kolber et al. 1994; Martin et al.
1994; Mills et al. 2004; Teeling et al. 2012). Additionally, major efforts have been
dedicated to understand the mechanisms that trigger phytoplankton blooms and the
observed temporal and spatial phytoplankton species successions found in the ocean (Du
and Peterson 2001; Leblanc et al. 2009; Mahadevan et al. 2012; Tai and Palenik 2009;
Venrick 2009). Relatively few studies have examined the importance of coenzymes, such
as B-vitamins, in regulating phytoplankton community composition and growth in marine
systems (Bertrand et al. 2012; Bertrand et al. 2007; Carini et al. 2014; Gobler et al. 2007;
Koch et al. 2012; Koch et al. 2011; Koch et al. 2013; Paerl et al. 2015; Panzeca et al.
2008; Panzeca et al. 2006), despite the pioneering work, carried out decades ago by
101
Carlucci, Droop, Guillard, Provasoli, and Swift, who established the importance of
exogenous B-vitamins as essential growth factors for many algal species (Croft et al.
2005; Croft et al. 2006; Provasoli and Carlucci 1974; Sañudo-Wilhelmy et al. 2014). The
causes of phytoplankton blooms and species successions in the ocean, which are usually
dominated by one or a few phytoplankton species, cannot be explained entirely by
physical parameters and the availability of inorganic macro- and micronutrients alone
(Guillard 1968). For example, changes in environmental factors alone cannot easily
explain a temporal succession of phytoplankton species within the same genera (Guillard
1968). B-vitamins have only been sparsely studied and their requirements are
paraphyletic and variable within taxonomic groups (Croft et al. 2006; Gobler et al. 2007;
Koch et al. 2011; Panzeca et al. 2006; Sañudo-Wilhelmy et al. 2014); therefore, differing
B-vitamin requirements may potentially explain species succession within a bloom.
B-vitamin availability can limit the growth of phytoplankton species that
constitute a dominant component of the biological carbon pump in aquatic ecosystems
(Bertrand et al. 2007; Panzeca et al. 2006; Sañudo-Wilhelmy et al. 2014) because they
facilitate a number of key enzymatic reactions in essential biochemical pathways,
including: the Calvin cycle, Krebs cycle, and a host of other reactions (Monteverde et al.
2017; Sañudo-Wilhelmy et al. 2014). Despite the crucial role that B-vitamins play in
cellular metabolism, many phytoplankton and marine bacterioplankton are B-vitamin
auxotrophs (obligate requirement), and are unable to synthesize the B-vitamins thiamin
(B
1
), biotin (B
7
), and cobalamin (B
12
) de novo (Carini et al. 2013; Carlucci and Silberna
1969; Giovannoni et al. 2005; Haines and Guillard 1974; Provasoli 1963; Provasoli and
Carlucci 1974; Swift 1980). Additionally, it has recently been demonstrated that many
102
microbial species require separate biochemical congeners and biosynthetic precursors of
these vitamins in order to meet their metabolic vitamin demands (Carini et al. 2014; Heal
et al. 2016; Helliwell et al. 2016; Paerl et al. 2015; Paerl et al. 2016). Therefore, in order
to gain a complete understanding of B-vitamin requirements, multiple B-vitamin
congeners must be studied. These include three forms of thiamin (B
1
, TMP, TPP), a
precursor moiety of thiamin (HMP), four forms of cobalamin with different upper axial
ligands (bioactive: AB
12
, MB
12
; bioinactive: CB
12
, HB
12
), and the amino acid methionine.
A few recent studies have begun to implicate B-vitamins as key factors regulating the
growth of auxotrophic phytoplankton species, and therefore species competition in situ
(Koch et al. 2011; Provasoli and Carlucci 1974; Sanudo-Wilhelmy et al. 2006; Tang et al.
2010). However, no field study has been conducted on the ecological relevance of B-
vitamins in a highly productive upwelling region, despite the recognized importance of
these regions for global primary production and biogeochemical cycles (Capone and
Hutchins 2013).
In order to assess the impact of B-vitamins on planktonic bloom succession, and
to simultaneously study the differential B-vitamin requirements of the microbial
community, we conducted several B-vitamin grow-out mesocosm experiments in a
coastal upwelling system within the Southern California Bight at the San Pedro Ocean
Time series (SPOT). The dynamics of the seasonal phytoplankton and bacterioplankton
species succession has been well characterized in the Southern California Bight
ecosystem (Chow et al. 2013; Countway et al. 2010; Du and Peterson 2001; Hu et al.
2016; Needham and Fuhrman 2016; Schnetzer et al. 2007; Venrick 2009). The observed
seasonal shifts in the phytoplankton abundance and production in this region are
103
controlled by seasonal upwelling occurring in the spring, followed by its relaxation
leading to stratification in the summer and fall, and finally deep mixing by winter storms
(Hickey 1992). However, the factors that control which specific species of phytoplankton
blooms are poorly understood, and likely include B-vitamin availability (Koch et al.
2011; Panzeca et al. 2006). The differential B-vitamin requirements and synthesis
capabilities of each organism have the potential to greatly change the ability of one
organism to dominate over another, depending on the availability of exogenous B-
vitamins in the environment (Sañudo-Wilhelmy et al. 2014). Based on the previous
evidence, we hypothesized that co-limitation by B-vitamins and macronutrients exists,
and that the additions of these growth factors would cause dramatic shifts in the
community structure, species succession, and growth rate. Never before have direct
environmental particulate B-vitamin quotas been measured in a mesocosm experiment.
Therefore the metabolic fate of the added B-vitamins is largely unknown. By applying
the recently developed LCMS based particulate B-vitamin analytical method (Suffridge
et al. 2017a), it is possible, for the first time, to measure the intracellular quotas of the
different chemical forms of the B-vitamins during the enrichment in an environmentally
relevant phytoplankton community. Using this technique we are able to address
questions about the changing B-vitamin requirements based on growth rates, and the
variable B-vitamin requirements of distinct microbial groups. This information is vital to
further the understanding of how B-vitamins influence microbial growth and community
composition.
104
Methods 4.3
4.3.1 Sample collection
Samples were collected at the San Pedro Ocean Time series (SPOT) station (33°
32.87’N 118°23.59’W) which is located in the Southern California Bight between the
port of Los Angeles and Catalina Island. Sampling was conducted at local apparent noon
on March 12th, 2015 aboard the R/V Yellowfin as a part of the monthly SPOT Program
and the USC Microbial Observatory. Due to the 2015 El Niño event, the typical seasonal
upwelling at SPOT did not occur before our study, therefore the conditions present were
much more typical of the oligotrophic conditions observed in the Autumn (Chow et al.
2013; Cram et al. 2015; Needham and Fuhrman 2016). Bulk seawater was collected
using a high volume, acid washed, hand powered pump from a depth of 3m. 400L of
seawater was stored in acid washed 20L cubitainers. Cubitainers were stored in the shade
and were kept at ambient seawater temp (17.2C) using the ship’s flow through seawater
system. Samples were then transported to USC.
4.3.2 Incubation setup
Bulk seawater stored in 20L cubitainers was homogenized and transferred into the
18, 10L experimental cubitainers. Six treatments were conducted with biological
triplicates: no addition control, nutrients (20µM NaNO
3
, 2µM NaH
2
PO
4
, 20µM SiOH
4
),
nutrients + B
1
(300pM), nutrients + B
7
(100pM), nutrients + B
12
(100pM), and nutrients
+B
1
+B
7
+B
12
. The concentrations given are the final concentration in the 10L
experimental bottle, and they were chosen to match upwelling conditions at SPOT (Chow
et al. 2013; Cram et al. 2015; Needham and Fuhrman 2016; Sañudo-Wilhelmy et al.
2012). Bottles were incubated in a temperature controlled cold room on USC’s main
105
campus set for ambient conditions at SPOT (17.2°C). White light was provided at 250µE
with a light-dark cycle mimicking the ambient conditions. Due to the remarkably low
biomass initially present, there was an exceptionally long lag phase. Growth was first
apparent on the 5th day. Additional B-vitamins were added at this point (150pM B
1
,
50pM B
7
, and 50pM B
12
) to compensate for B-vitamin degradation.
4.3.3 Experimental sampling
Two types of sampling were conducted during the experiments: Basic and Full.
All sampling occurred at the same time daily. Basic sampling occurred daily to assess
growth via in vivo fluorescence and flow cytometry cell counts. Full sampling occurred
initially, once during exponential growth (Tex), and once during stationary phase (Tst).
At full sampling points samples for particulate and dissolved B-vitamins, eukaryotic
microscopic diversity, POC/N/P, and extracted size fractionated Chlorophyll A (Chl-a)
were taken from each bottle.
4.3.4 B-vitamin sample collection
Samples for dissolved and particulate B-vitamins were collected as has previously
been described (Suffridge et al. 2017a). 1.5L of sample was filtered using a methanol
cleaned, seawater rinsed paristaltic filtration apparatus. In some cases less volume was
filtered as the increased cellular density clogged the filters. The suspended particles were
collected with two serial, in-line filters: a 3µm pore size, 47mm diameter poly carbonate
filter (Nucleopore) followed by a 0.2µm pore size, 47mm diameter Supor filter (Pall
Biosciences). The 0.2µm-3µm size fraction was designed to capture the picoplankton (PP
hereafter) including bacterioplankton, cyanobacteria, and picoeukaryotes (Sherr and
Sherr 2008). The ≥3µm fraction was designed to capture nanoplankton and
106
microplankton (NP hereafter) which include the eukaryotic phytoplankton such as
diatoms and dinoflagelletes (Sherr and Sherr 2008). Particulate samples were transferred
into cryovials and frozen at -80°C until analysis. The filtrate was collected in methanol
cleaned, seawater rinsed, amber HDPE bottles for dissolved-pool analysis. Dissolved
samples were frozen at -20°C until analysis.
4.3.5 B-vitamin analysis
B-vitamin samples were analyzed as has been previously described (Suffridge et
al. 2017a). Both dissolved and particulate pools of nine B-vitamin congeners were
measured; they include: unphosphorylated thiamin (B
1
), thiamin monophosphate (TMP),
thiamin pyrophosphate (TPP), a thiamin precursor moiety (4-amino-2-methyl- 5-
pyrimidinyl methanol, HMP), biotin (B
7
), adenosylcobalamin (AB
12
), methylcobalamin
(MB
12
), hydroxycobalamin (HB
12
), and cyanocobalamin (CB
12
). Additionally the amino
acid methionine (Met) was also measured. In order to assess the total available thiamin
and cobalamin pools we calculated total thiamin (tB
1
) and total cobalamin (tB
12
) by
summing their respective congers (B
1
, TMP and TPP for tB
1
; AB
12
, MB
12
, HB
12
, CB
12
for
tB
12
). Dissolved samples were preconcentrated by passing the sample over a C
18
resin at
two pHs (6.5 and 2.0), followed by elution into 12ml of methanol. A nitrogen dryer was
used to evaporate the samples to 250µl, providing a concentration factor of six orders of
magnitude. Particulate B-vitamins were extracted from the filters by bead beating in 3ml
of acidic methanol lysis solution followed by a chloroform liquid phase extraction to
reduce signal suppression (Suffridge et al. 2017a). Analysis of both dissolved and
particulate B-vitamin samples was conducted using a Thermo Scientific Quantum Access
electrospray ionization triple quadrupole mass spectrometer, coupled to a Thermo
107
Scientific Accela High Speed Liquid Chromatography (LC/MS) system (Suffridge et al.
2017a). The LC system used a stable- bond C
18
reversed-phase column (Discovery HS
C
18
10 cm × 2.1mm, 5µm column, Supelco Analytical), with a methanol:water gradient
program (Suffridge et al. 2017a). Quantification was conducted using two stable
isotopically labeled internal standards (thiamin and riboflavin) (Suffridge et al. 2017a).
4.3.6 Heterotrophic bacteria and picoautotroph quantification
Samples for flow cytometry were collected, fixed with 2% formalin, and frozen at
-80°C. Analysis for the cellular abundance of heterotrophic bacteria, Synechococcus, and
picoeukaryotes was conducted using a BD Accuri C6 flow cytometer (Becton Dickerson
and Company).
4.3.7 Eukaryotic plankton diversity
Samples (50ml volume) were collected from each replicate of six treatments and
preserved at 4°C in the dark with the addition of acidified Lugol’s solution and
enumerated using an Accu-Scope 3032 inverted microscope.
4.3.8 Specific growth rates
Specific growth rates (SGR; µ) were calculated based on the daily in vivo
fluorescence readings, heterotrophic bacterial abundance, and picoautotroph
(Synechococcus and picoeukaryotes) abundance. The formula µ= ln(N
t2
/ N
t1
)/(t
2
-t
1
) was
used, where N
t1
, and N
t2
refer to biomass at time 1 (t
1
)and 2 (t
2
) (in days) respectively
(Ihnken et al. 2011).
108
4.3.9 Chlorophyll A (Chl-a) quantification
In vivo chlorophyll measurements were taken daily to assess phytoplankton
growth. 5ml of each experimental treatment were directly analyzed with a Turner
Designs 10-AU
TM
fluorometer to determine the in vivo relative Chl-a fluorescence (Fu et
al. 2007). While the relative fluorescence units (RFU) of in vivo Chl-a cannot be
converted to concentration units, we observed a strong significant correlation between in
vivo chlorophyll and extracted chlorophyll during this study (Pearson’s r=0.94, p-
value<0.001), indicating that this measurement can be used as a reliable daily proxy for
extracted chlorophyll concentration (Fu et al. 2007).
Chl-a concentrations were measured initially and at Tex and Tst. A sample
volume of 40ml from each replicate was filtered through GF/F glass fiber filters, 3.0µm,
and 8.0µm polycarbonate membrane filters for size fractionated Chl-a analyses. After
adding 6ml of 90% acetone, Chl-a was extracted in the freezer at -20°C and measured
using the non-acidification method with a Turner Designs 10-AU
TM
fluorometer after 24
h (Welschmeyer 1994).
4.3.10 Elemental (C, N, P) quantification
Elemental ratios were obtained by measuring particulate organic carbon and
nitrogen, and phosphorus (POC, PON, and POP). For particulate organic carbon and
nitrogen (POC and PON), 100ml was filtered onto pre-combusted (500°C, 2h) GF/F
filters, which were then wrapped in aluminum foil and dried at 55°C. POC and PON were
analyzed on a Costech Elemental Analyzer using methionine and acetanilide as
references to calibrate the system at the beginning of the measurements (Fu et al. 2007).
109
For particulate organic phosphorus (POP) samples, 40ml was filtered onto pre-
combusted (500°C, 2h) GF/F filters and rinsed twice with 2ml 0.17M Na
2
SO
4
solution.
The filters were placed in 20ml borosilicate scintillation vials (pre-combusted at 500°C,
overnight) to which was added 2ml 0.017M MgSO
4
solution. The vials were then
covered with aluminum foil and dried at 95°C, followed by combustion at 450-500°C for
2h. After cooling to room temperature, 5ml of 0.2M HCl solution was added to each vial,
which were then tightly capped and heated at 80°C for thirty minutes to digest POP into
inorganic phosphate. The standard molybdate colorimetric method was used to analyze
the samples (Solorzano and Sharp 1980). Three GF/F filters were treated in the same way
as the samples for blank determinations.
4.3.11 Statistical analysis
All data was analyzed, and all statistical analysis was conducted using the
software environment R (R Core Team 2015). All figures were created using the ggplot2
software package for R (Wickham 2009). Particulate B-vitamin mean concentrations
were calculated by taking the average of the B-vitamin concentrations from each
biological replicate. Particulate B-vitamin quotas were calculated for each biological
replicate, and the mean quota values presented are the average of the normalized
biological replicates. The standard deviations presented for both concentrations and
quotas represent the observed biological variability.
110
Results and Discussion 4.4
4.4.1 Background conditions at SPOT
The oceanographic conditions at SPOT were uncharacteristically oligotrophic
during our sampling in March 2015. SPOT is known to have strong seasonality, with
dominate upwelling-induced phytoplankton blooms occurring between February and
May, followed by cyanobacteria dominated stratified oligotrophic conditions occurring in
summer and fall (Chow et al. 2013; Countway et al. 2010; Cram et al. 2015; Haskell et al.
2016; Needham and Fuhrman 2016; Steele et al. 2011). The 2015 El Niño event
interrupted the upwelling, resulting in conditions much more similar to the oligotrophic
conditions typically found in late summer and fall (Chow et al. 2013; Countway et al.
2010; Cram et al. 2015; Cram et al. 2016; Haskell et al. 2016; Needham and Fuhrman
2016). The dissolved phosphate concentration at 3m was observed to be 0.25µM, which
is lower than the variability observed in March over 10 years between 2000 and 2011
which ranged from 0.3-0.8µM (Chow et al. 2013). The observed phosphate concentration
was an order of magnitude lower than the concentration that was added in this experiment
(2µM). The Chl-a concentration that we observed at SPOT (0.1µg/L, Table 4.1 and
Figure 4.2) was about half of the 10 year average concentration (0.25µg/L) from the
surface at SPOT, and the average DCM depth for March was 30m, compared to the 40m
DCM that was observed in March 2015 (Chow et al. 2013). The observed bacterial
abundance (1.3 x10
6
cells/ml, Table 4.2, Figure 4.1) fell below the range of variability
(1.5 x10
6
-2.5 x10
6
cells/ml) reported between 2000 and 2011 (Chow et al. 2013). The
observed POP concentration was 0.01µM and the particulate C:N:P ratio was 580:35:1
suggesting that phosphorus was the limiting nutrient at SPOT during our sampling (Table
111
4.1 and Figure 4.2). Taken together, the observed nutrient and biomass parameters
indicate that biomass at SPOT during our sampling was lower than what is typical for this
location (Figure 4.1 and Figure 4.2).
112
Table 4.1 Daily Biomass Measurements. Abundances (cells/ml) of heterotrophic bacteria, picoeukaryotes,
and Synechococcus were measured daily using a flow cytometer. In vivo Chl-a fluorescence was also measured
daily. The exponential phase sample point (Tex) and the stationary phase sample point (Tst) are indicated. If
standard deviation is not listed, it indicates that only one reading was made (listed in the “mean” column).
Treatment Date mean St. Dev. mean St. Dev. mean St. Dev. mean St. Dev.
Control 3/13 1.30E+06 0.01 0.00 2.00E+03 1.20E+04
Control 3/14 1.55E+06 0.01 0.00 4.00E+03 1.70E+04
Control 3/15 1.98E+06 0.01 0.00 6.00E+03 3.60E+04
Control 3/16 1.79E+06 0.02 0.00 6.00E+03 1.00E+04
Control 3/17 9.82E+05 0.02 0.00 9.00E+03 5.00E+03
Control 3/18 0.02 0.00
Control 3/19 5.07E+05 0.01 0.00 4.00E+03 2.00E+03
Control 3/20 Tex 6.25E+05 0.01 0.00 2.00E+03 2.00E+03
Control 3/21 8.66E+05 0.01 0.00 2.00E+03 2.00E+03
Control 3/22 1.04E+06 0.01 0.00 1.00E+03 1.00E+03
Control 3/23 1.24E+06 0.01 0.00 1.00E+03 1.00E+03
Control 3/24 Tst 1.38E+06 0.01 0.00 1.00E+03 2.00E+03
Control 3/25 1.53E+06 0.01 0.01 2.00E+03 2.00E+03
N 3/13 1.33E+06 4.38E+04 0.01 0.00 2.50E+03 7.07E+02 9.60E+03 8.49E+02
N 3/14 1.44E+06 7.14E+04 0.01 0.00 4.00E+03 0.00E+00 1.48E+04 1.06E+03
N 3/15 1.38E+06 1.20E+04 0.01 0.00 8.00E+03 1.41E+03 2.84E+04 4.74E+03
N 3/16 1.17E+06 6.36E+03 0.02 0.00 1.60E+04 2.83E+03 4.18E+04 3.89E+03
N 3/17 8.32E+05 1.70E+04 0.04 0.00 2.60E+04 8.49E+03 4.17E+04 3.75E+03
N 3/18 6.33E+05 0.07 0.01 2.20E+04 3.30E+04
N 3/19 8.76E+05 2.83E+03 0.13 0.04 3.25E+04 1.48E+04 3.80E+04 7.07E+01
N 3/20 Tex 2.23E+06 1.04E+05 0.26 0.06 4.10E+04 1.27E+04 6.72E+04 5.37E+03
N 3/21 4.42E+06 2.99E+05 0.58 0.06 6.50E+04 2.26E+04 1.27E+05 1.77E+04
N 3/22 5.92E+06 1.49E+06 0.85 0.21 6.65E+04 9.19E+03 2.12E+05 5.59E+04
N 3/23 7.32E+06 2.27E+06 0.86 0.19 5.05E+04 4.95E+03 1.85E+05 6.86E+04
N 3/24 7.54E+06 2.71E+06 0.61 0.08 4.10E+04 1.70E+04 9.65E+04 2.47E+04
N 3/25 Tst 9.58E+06 1.19E+05 0.56 0.03 4.60E+04 2.69E+04 5.45E+04 3.03E+04
B 1 3/13 1.31E+06 0.01 0.00 2.00E+03 1.00E+04
B 1 3/14 1.53E+06 0.01 0.00 3.00E+03 1.50E+04
B 1 3/15 1.96E+06 0.01 0.00 6.00E+03 3.70E+04
B 1 3/16 1.88E+06 0.02 0.00 1.70E+04 6.90E+04
B 1 3/17 1.33E+06 0.04 0.00 4.10E+04 6.50E+04
B 1 3/18 8.75E+05 0.07 0.01 6.50E+04 4.00E+04
B 1 3/19 1.28E+06 0.12 0.05 6.80E+04 4.70E+04
B 1 3/20 Tex 2.14E+06 0.34 0.13 7.85E+04 5.30E+04 1.01E+05 2.33E+04
B 1 3/21 3.89E+06 0.57 0.09 9.10E+04 1.45E+05
B 1 3/22 4.67E+06 0.77 0.15 6.45E+04 2.33E+04 1.88E+05 1.34E+05
B 1 3/23 5.28E+06 0.51 0.27 6.10E+04 2.26E+04 5.20E+04 5.66E+03
B 1 3/24 Tst 5.75E+06 0.37 0.17 4.90E+04 3.11E+04 4.20E+04 4.24E+03
B 1 3/25 7.17E+06 0.33 0.15 5.50E+04 4.24E+04 2.75E+04 4.95E+03
B 7 3/13 1.29E+06 0.01 0.00 2.00E+03 1.00E+04
B 7 3/14 1.42E+06 0.01 0.00 4.00E+03 1.50E+04
B 7 3/15 1.36E+06 0.01 0.00 7.00E+03 2.70E+04
B 7 3/16 1.15E+06 0.02 0.00 1.20E+04 3.90E+04
B 7 3/17 8.90E+05 0.04 0.00 1.80E+04 3.70E+04
B 7 3/18 6.47E+05 0.07 0.00 1.90E+04 2.80E+04
B 7 3/19 1.06E+06 0.10 0.02 1.90E+04 3.40E+04
B 7 3/20 Tex 2.62E+06 0.25 0.09 3.15E+04 7.78E+03 7.30E+04 9.90E+03
B 7 3/21 4.86E+06 0.52 0.18 4.30E+04 1.30E+05
B 7 3/22 6.57E+06 0.67 0.11 4.85E+04 6.36E+03 2.30E+05 4.95E+03
B 7 3/23 6.66E+06 0.71 0.05 4.00E+04 8.49E+03 1.11E+05 2.12E+03
B 7 3/24 5.80E+06 0.59 0.02 3.35E+04 7.07E+02 7.50E+04 4.53E+04
B 7 3/25 Tst 4.90E+06 0.53 0.05 4.90E+04 4.24E+03 2.90E+04 1.41E+03
B 12 3/13 1.28E+06 0.01 0.00 2.00E+03 2.20E+04
B 12 3/14 1.30E+06 0.01 0.00 4.00E+03 1.50E+04
B 12 3/15 1.24E+06 0.01 0.00 8.00E+03 2.60E+04
B 12 3/16 1.21E+06 0.02 0.00 1.60E+04 3.70E+04
B 12 3/17 8.71E+05 0.04 0.01 2.50E+04 4.10E+04
B 12 3/18 0.10 0.04 3.60E+04 4.20E+04
B 12 3/19 9.33E+05 0.35 0.19 3.70E+04 5.10E+04
B 12 3/20 Tex 1.64E+06 1.02 0.34 3.90E+04 1.70E+04 7.05E+04 4.95E+03
B 12 3/21 3.55E+06 1.25 0.09 4.30E+04 9.90E+04
B 12 3/22 5.74E+06 1.06 0.08 4.20E+04 2.83E+03 3.95E+04 9.19E+03
B 12 3/23 Tst 5.02E+06 0.91 0.08 4.30E+04 4.24E+03 1.75E+04 1.06E+04
B 12 3/24 6.25E+06 0.72 0.08 5.40E+04 5.66E+03 1.40E+04 1.13E+04
B 12 3/25 6.58E+06 0.61 0.06 4.50E+04 1.41E+03 7.00E+03 7.07E+03
All 3/13 1.29E+06 0.01 0.00 2.00E+03 1.00E+04
All 3/14 1.36E+06 0.01 0.00 4.00E+03 1.40E+04
All 3/15 1.30E+06 0.01 0.00 7.00E+03 2.60E+04
All 3/16 1.08E+06 0.03 0.00 1.40E+04 3.70E+04
All 3/17 7.56E+05 0.05 0.01 2.30E+04 3.80E+04
All 3/18 5.61E+05 0.11 0.02 3.10E+04 3.10E+04
All 3/19 7.94E+05 0.36 0.08 4.00E+04 3.60E+04
All 3/20 Tex 2.46E+06 1.06E+05 1.02 0.20 6.45E+04 2.33E+04 5.10E+04 9.90E+03
All 3/21 4.20E+06 6.22E+05 1.16 0.07 5.10E+04 8.85E+04 9.19E+03
All 3/22 4.85E+06 1.20E+06 0.92 0.09 6.50E+04 1.13E+04 1.75E+04 7.07E+02
All 3/23 Tst 3.75E+06 1.49E+06 0.75 0.04 3.55E+04 1.63E+04 1.35E+04 2.12E+03
All 3/24 4.90E+06 1.11E+06 0.63 0.07 4.15E+04 1.06E+04 1.10E+04 0.00E+00
All 3/25 5.04E+06 0.56 0.05 4.10E+04 1.41E+03 1.00E+04
Heterotrophic
Bacterial Abundance
(cells/ml) In vivo Chl-a
Picoeukaryote
Abundance (cells/ml)
Synechococcus
Abundance (cells/ml) Time
Point
113
Figure 4.1 Daily Biomass Measurements. Abundances (cells/ml) of heterotrophic bacteria, picoeukaryotes,
and Synechococcus were measured daily using a flow cytometer. In vivo Chl-a fluorescence was also measured
daily. The exponential phase sample point (Tex) is indicated by squares. The stationary phase sample point
(Tst) is indicated by triangles.
2.5e+06
5.0e+06
7.5e+06
1.0e+07
0.0
0.5
1.0
0e+00
5e+04
1e+05
0e+00
1e+05
2e+05
3e+05
Mar 14
Mar 16
Mar 18
Mar 20
Mar 22
Mar 24
Control
N
B
1
B
7
B
12
All
Heterotrophic Bacterial Abundance
In Vivo Chl-a
Picoeukaryote Abundance
Synechococcus Abundance
cells/ml RFU cells/ml cells/ml
FIGURE 3.1
114
Figure 4.2 Biomass Measurements at the B-vitamin sample points. Extracted Chl-a concentrations (µg/L)
were determined from three size fractions: 0.7-3µm, ≥3µm, and total (GF/F). Concentrations of particulate
organic carbon (POC), nitrogen (PON), and phosphorus (POP) were also measured (µM). Measurements
occurred at the initial samples, the exponential phase sample point (Tex) and the stationary phase sample point
(Tst). The abundance (cells/ml) of heterotrophic bacteria, Synechococcus, and picoeukaryotes are (also
presented in Figure 4.1) are plotted here at the two vitamin sample points.
0
5
10
0
2
4
6
0.0
2.5
5.0
7.5
10.0
2.5e+06
5.0e+06
7.5e+06
1.0e+07
0
25000
50000
75000
100000
0
20000
40000
60000
80000
0
100
200
300
0
5
10
15
20
0.0
0.5
1.0
1.5
Mar 13
Mar 15
Mar 17
Mar 19
Mar 21
Mar 23
Mar 13
Mar 15
Mar 17
Mar 19
Mar 21
Mar 23
Mar 13
Mar 15
Mar 17
Mar 19
Mar 21
Mar 23
Total Chl-a 0.7-3μm Chl-a ≥3μm Chl-a
μg Chl-a / L
cells/ml
Heterotrophic Bacteria Picoeukaryote Synechococcus
POC POP PON
μM
Control
N
B
1
B
7
B
12
All
FIGURE 3.S1
115
Figure 4.3 Daily specific growth rate. Calculations were made for heterotrophic bacteria, Synechococcus,
picoeukaryotes, and in vivo Chl-a based off the abundances in Figure 1 and Table 4.1. The exponential phase
sample point (Tex) is indicated by squares. The stationary phase sample point (Tst) is indicated by triangles.
−0.5
0.0
0.5
1.0
−0.5
0.0
0.5
1.0
−0.5
0.0
0.5
1.0
−1
0
1
Mar 14
Mar 16
Mar 18
Mar 20
Mar 22
Mar 24
Specific Growth Rate
Heterotrophic Bacterial Abundance
In Vivo Chl-a
Picoeukaryote Abundance
Synechococcus Abundance
Control
N
B
1
B
7
B
12
All
116
Table 4.2 Biomass Measurements. Extracted Chl-a concentrations (µg/L) were determined from three size
fractions: 0.7-3µm, ≥3µm, and total (GF/F). Concentrations of particulate organic carbon (POC), nitrogen
(PON), and phosphorus (POP) were also measured (µM). Measurements occurred at the initial samples, the
exponential phase sample point (Tex) and the stationary phase sample point (Tst). If standard deviation is not
listed, it indicates that only one reading was made (listed in the “mean” column).
Treatment Timepoint mean st. dev. mean st. dev. mean st. dev. mean st. dev. mean st. dev. mean st. dev.
Control T0 0.133 0.0447 0.177 6.38 0.387 0.0110
Control Tex 0.294 0.0148 0.203 0.0562 0.497 0.0711 14.9 2.08 1.69 0.0149 0.0473 0.0178
Control Tst 0.143 0.0315 0.0737 0.0022 0.217 0.0293 15.9 2.88 1.72 0.0053 0.0536
N Tex 1.69 0.562 3.19 0.286 4.88 0.849 45.1 7.77 6.06 1.16 0.224 0.0089
N Tst 1.42 1.24 6.65 1.41 8.07 0.170 248 18.6 19.0 0.740 1.20 0.0223
B
1
Tex 2.05 0.86 4.86 1.87 6.91 2.73 55.9 8.79 7.46 1.89 0.318 0.0892
B
1
Tst 2.59 0.159 5.69 2.07 8.28 1.91 251 8.09 18.8 0.912 1.20 0.0178
B
7
Tex 1.47 1.22 3.98 0.11 5.45 1.34 56.6 9.70 6.60 0.0083 0.255 0.0089
B
7
Tst 1.94 0.233 4.61 0.0212 6.54 0.212 251 4.87 19.0 1.25 1.28 0.0089
B
12
Tex 5.12 1.17 7.86 2.39 13.0 1.22 93.4 25.2 13.0 3.99 0.602 0.232
B
12
Tst 4.19 2.29 5.67 0.488 9.86 1.80 325 30.4 21.4 1.07 1.49 0.223
All Tex 5.92 0.392 5.77 1.67 11.7 2.06 86.1 13.7 13.3 0.491 0.498 0.129
All Tst 4.28 1.73 4.82 0.477 9.11 1.25 300 39.3 20.6 0.295 1.43 0.187
POP (µM)
Chl-a 0.7-
3µm (µg/L)
Chl-a ≥3µm
(µg/L)
Total Chl-a
(µg/L) POC (µM) PON (µM)
117
Dissolved B-vitamin concentrations were measured at the surface at SPOT to
determine the background dissolved vitamin levels. The concentrations of the thiamin
congeners were 1.0±0.2pM B
1
, 0.45±0.15pM TMP, and B.D.L. TPP (<0.1pM). The
concentration of the thiamin precursor HMP in the ambient SPOT water was 9.6±3.3pM.
Vitamin B
7
had the highest observed dissolved concentration of the B-vitamins at
57.5±10.5pM. The amino acid methionine was below our detection limits (<4.0pM pM).
The concentrations of the cobalamin congeners (pM) were 0.27±0.05 CB
12
, 0.29±0.01
HB
12
, 0.42±0.2 AB
12
, and 0.85±0.03 MB
12
. The concentrations for all analytes are at the
low end of the previously reported ranges for these compounds in the ocean (Heal et al.
2014; Sañudo-Wilhelmy et al. 2012; Suffridge et al. 2017b). We observed dissolved
MB
12
to be the most abundant cobalamin congener, which is consistent with observations
from other regions of the ocean (Suárez-Suárez et al. 2011; Suffridge et al. 2017b). The
relative abundance of dissolved thiamin congeners, where B
1
is the most abundant
chemical form of this vitamin, is also consistent with previous results from other
oceanographic regions (Suffridge et al. 2017b). The dissolved tB
1
and tB
12
concentrations were between one and two orders of magnitude lower than the
concentrations that were added to our experiments (300pM B
1
as thiamin, 100pM B
12
as
cyanocobalamin). However, the dissolved B
7
concentration (57.5±10.5pM) was roughly
half the concentration that was added to the experiment (100pM). This indicates that
there was a substantial excess of dissolved B
1
and B
12
relative to their environmental
availability.
118
4.4.2 B-Vitamin Co-Limitation
Differential experimental responses to nutrient and B-vitamin additions suggest
that not only were biomass and nutrients low at SPOT, but that the phytoplankton
community was co-limited by both, inorganic nutrients and organic growth factors
availability (e.g., vitamin B
12
; Figure 4.1). Both Chl-a standing stock (both as in-vivo
Chl-a and extracted Chl-a) and Chl-a based specific growth rate (SGR) measurements
were observed to be highest in treatments containing nutrients and vitamin B
12
(Figure
4.1, Figure 4.2, and Figure 4.3; Table 4.1 and Table 4.2). At Tex the in vivo Chl-a values
for the B
12
and All treatments (1.0±0.3 B
12
, 1.0±0.2 All) were three times higher than the
biomass measured in the nutrients, B
1
, and B
7
treatments (0.26±0.06 nutrients, 0.34±0.13
B
1
, 0.25±0.1 B
7
). The control in vivo Chl-a was 0.008±0.003, which was significantly
lower (p<0.05) than the biomass observed in any of the nutrient addition treatments
(Figure 4.1). At Tst, all in vivo Chl-a readings from treatments containing nutrients
converged at 0.62±0.21 RFU (all nutrient treatments combined), and no significant
difference between the initial and final control in vivo Chl-a was observed (Figure 4.1).
At Tex, the B
12
and the All treatment had mean extracted Chl-a concentration of 12.9±1.2
µg/L and 11.7±2.1 µg/L respectively. The nutrients, B
1
, and B
7
treatments were
substantially lower with mean Chl-a concentrations of 4.8±0.85, 6.9±2.7, and 5.4±1.3
µg/L respectively. The B
12
containing treatments were also observed to have the maximal
Chl-a -based SGRs, with values of 1.24 B
12
and 1.19 All (Figure 4.3). These values,
which occurred the day prior to Tex (Figure 4.3), were double than those observed for the
nutrients, B
1
, and B
7
treatments (0.61,0.52, and 0.44 respectively) at the same time point.
The observed Chl-a concentrations at Tex were between one and two orders of
magnitude higher than the initial Chl-a concentrations (0.1 µg/L), providing strong
119
evidence that the initial community was limited by inorganic nutrients (N and P). The
two-fold increase in Chl-a concentrations and SGR in the treatments containing B
12
relative to those containing only inorganic nutrients suggests that the phytoplankton
community was co-limited by B
12
(Figure 4.1, Figure 4.2, and Figure 4.3). It has been
established that B
12
auxotrophy is present in a majority of phytoplankton (Croft et al.
2006; Sañudo-Wilhelmy et al. 2014) Additionally, our results are consistent with
previously studies that have demonstrated B
12
-colimitation in various groups of
phytoplankton (Bertrand et al. 2012; Bertrand et al. 2015; Bertrand et al. 2011; Koch et
al. 2012; Koch et al. 2011; Koch et al. 2013).
In contrast to the effect of B
12
and nutrient amendments on total community
biomass (as bulk Chl-a concentrations), picoeukaryote abundance indicated potential co-
limitation by nutrients and vitamin B
1
(Figure 4.1 and Figure 4.2). We observed a clear
increase in picoeukaryote abundance in the B
1
treatment relative to the other nutrient
containing treatments beginning three days prior to Tex (Figure 4.1). The SGR of
picoeukaryotes at this point, 1.04, was also the highest observed picoeukaryote SGR in
any treatment, and was about double the growth rates from the other nutrient containing
treatments (0.53-0.69; Figure 4.3). The maximum picoeukaryote abundance, 9.1x10
4
cells/ml, was observed in the B
1
treatment the day after Tex (Figure 4.1). The
picoeukaryote abundance in all other nutrient containing treatments overlapped for the
entire experiment, and at the point of maximum picoeukaryote abundance they ranged
from 4.3x10
4
in the B
12
treatment to 6.5x10
4
in the nutrient treatment (Figure 4.1). The
control treatment always had an order of magnitude lower cellular abundance (cells/ml)
than the next lowest treatment (Figure 4.1). These observations are consistent with the
120
widespread thiamin auxotrophy found in Prymnesiophytes, a class of picoeukaryotes
which are known to be abundant in the California Current and at SPOT (Countway et al.
2010; Hu et al. 2016; Needham and Fuhrman 2016; Paerl et al. 2015; Sañudo-Wilhelmy
et al. 2014; Worden and Not 2008).
Synechococcus abundance did not reach its maximum until two days after the Tex
sample point, potentially indicating that prior to this point it was not able to effectively
compete with the larger, blooming, phytoplankton (Figure 4.1, Figure 4.4; Table 4.1,
Table 4.2). At this point, there was no clear differentiation between nutrients, B
1
, and B
7
treatments, with cellular abundance ranging 1.9x10
5
(B
1
) to 2.3x10
5
(B
7
). Interestingly,
both treatments including B
12
(All and B
12
) were an order of magnitude lower than the
other nutrient containing treatments with cellular abundances of 3.9x10
4
and 1.7x10
4
respectively (Figure 4.1). It is possible that the low cellular abundances observed in the
B
12
containing treatments with respect to the other nutrient containing treatments was due
to larger phytoplankton being stimulated early in the experiment by the B
12
addition and
depleting the available nutrients in these treatments (Figure 4.4). This effect was not
observed in the other nutrient containing treatments as the total Chl-a in these treatments
was about half of the maximum observed concentrations in the B
12
containing treatments
(Figure 4.1 and Figure 4.2). Alternatively, the low Synechococcus abundances in the B
12
containing treatments co-occurred with high abundances of the Pseudonitzschia, a known
HAB species (Figure 4.4); therefore, it is possible that domoic acid produced by
Pseudonitzschia inhibited Synechococcus growth. However, domoic acid was not
measured in this experiment, so it is not possible to determine if the Pseudonitzschia
were in fact producing this toxin.
121
Figure 4.4 Eukaryotic Plankton Diversity. Cellular abundance (cells/ml) of eukaryotic phytoplankton
(panel A) was determined via microscopy. Relative abundance of each group at each time point and treatment
(panel B) was calculated.
0
2
4
6
8
0
1000
2000
3000
4000
0
20000
40000
Control
N
N+B1
N+B7
N+B12
All
N
N+B1
N+B7
N+B12
All
cells/ml
Exponential Initial Stationary
0%
25%
50%
75%
100%
Control
N
N+B1
N+B7
N+B12
All
N
N+B1
N+B7
N+B12
All
Percent Abundance
Chaetocerous
Ciliate
Cylindrothecea
Other Diatoms
Dinoflagellate
Other
Pseudonitzschia
Exponential Initial Stationary
A.
B.
122
Table 4.3 Eukaryotic Plankton Diversity. Abundances of eukaryotic plankton were determined via cell
counts. The relative abundances of each group were calculated for each treatment and time point.
Treatment Time Point Organism
Mean
Abundance
(cells/ml) st.dev.
Relative
Abundance
Control T0 Chaetoceros 1.32 0.28 16%
Control T0 Ciliate 0.478 0.57 6%
Control T0 Cylindrotheca 0.00 0.00 0%
Control T0 Other Diatom 0.624 1.24 8%
Control T0 Dinoflagellate 5.60 3.04 69%
Control T0 Other 0.00 0.00 0%
Control T0 Pseudonitzschia 0.04 0.0212 0%
N Tex Chaetoceros 526 246 59%
N Tex Ciliate 3.33 2.36 0%
N Tex Other Diatom 2.78 0.962 0%
N Tex Dinoflagellate 135 4.41 15%
N Tex Pseudonitzschia 222 48.8 25%
N Tst Chaetoceros 10300 2000 27%
N Tst Cylindrotheca 1130 721 3%
N Tst Other Diatom 600 606 2%
N Tst Dinoflagellate 817 1070 2%
N Tst Pseudonitzschia 25800 4210 67%
B
1
Tex Chaetoceros 1130 903 80%
B
1
Tex Cylindrotheca 7.78 9.18 1%
B
1
Tex Other Diatom 5.67 5.73 0%
B
1
Tex Dinoflagellate 143 52.0 10%
B
1
Tex Pseudonitzschia 116 56.6 8%
B
1
Tst Chaetoceros 4950 2640 24%
B
1
Tst Cylindrotheca 1270 660 6%
B
1
Tst Other Diatom 350 609 2%
B
1
Tst Dinoflagellate 1330 1500 6%
B
1
Tst Pseudonitzschia 12700 12700 62%
B
7
Tex Chaetoceros 716 356 57%
B
7
Tex Ciliate 5.00 4.71 0%
B
7
Tex Cylindrotheca 8.89 8.55 1%
B
7
Tex Other Diatom 5.56 4.79 0%
B
7
Tex Dinoflagellate 182 6.74 15%
B
7
Tex Pseudonitzschia 334 84.2 27%
B
7
Tst Chaetoceros 18300 17800 35%
B
7
Tst Ciliate 88.9 38.5 0%
B
7
Tst Cylindrotheca 1500 1320 3%
B
7
Tst Other Diatom 587 615 1%
B
7
Tst Dinoflagellate 1960 1930 4%
B
7
Tst Pseudonitzschia 30300 13200 57%
B
12
Tex Chaetoceros 2290 2039 65%
B
12
Tex Ciliate 5.00 0.00 0%
B
12
Tex Cylindrotheca 20.0 21.2 1%
B
12
Tex Other Diatom 24.5 21.9 1%
B
12
Tex Dinoflagellate 289 196 8%
B
12
Tex Other 5.00 0.00 0%
B
12
Tex Other 7.50 3.54 0%
B
12
Tex Pseudonitzschia 895 1100 25%
B
12
Tst Chaetoceros 18000 7870 47%
B
12
Tst Cylindrotheca 2160 1020 6%
B
12
Tst Other Diatom 390 506 1%
B
12
Tst Dinoflagellate 1470 1320 4%
B
12
Tst Pseudonitzschia 16300 4110 43%
All Tex Chaetoceros 2880 743 68%
All Tex Ciliate 7.50 3.54 0%
All Tex Cylindrotheca 25.0 18.0 1%
All Tex Other Diatom 20.9 13.6 0%
All Tex Dinoflagellate 430 176 10%
All Tex Other 25.0 28.3 1%
All Tex Pseudonitzschia 847 452 20%
All Tst Chaetoceros 15100 3730 41%
All Tst Ciliate 83.3 23.6 0%
All Tst Cylindrotheca 1420 1250 4%
All Tst Other Diatom 396 406 1%
All Tst Dinoflagellate 1880 2080 5%
All Tst Pseudonitzschia 17500 6560 48%
123
Heterotrophic bacterial abundance was not co-limited by B-vitamins during this
study, and their bloom occurred about two days after phytoplankton growth was first
observed (Figure 4.1; Table 4.1), which is consistent with known patterns of planktonic
bloom succession at SPOT where bacterioplankton bloom only occur after phytoplankton
have created a sufficient supply of organic matter (Chow et al. 2013; Cram et al. 2015;
Needham and Fuhrman 2016). No significant differences between the heterotrophic
bacterial abundances were observed in the vitamin addition treatments relative to the
nutrient addition. However, the nutrient addition was significantly different from the
control results, suggesting limitation by inorganic nutrients (Figure 4.1). Bacterial
abundances were 1.3 x10
6
cells/ml at the initial time point. At the Tex the nutrient
treatment abundances were 2.2 x10
6
±1.0 x10
5
cells/ml, reaching 9.5x10
6
± 1.1 x10
5
cells/ml at stationary phase (Figure 4.1). The maximum observed bacterial SGRs for all
treatments containing nutrients occurred at the Tex where the highest SGR (1.13, All)
was double the lowest SGR (0.51 B
1
; Figure 4.3). Bacterioplankton are known to include
both B-vitamin producers and auxotrophs (Sañudo-Wilhelmy et al. 2014), and it has
recently been demonstrated, using transcriptomics that there are strong B-vitamin
mediated relationships between different groups of bacterioplankton (Gomez-Consarnau
et al. 2017). While we do not have the data to assess the bacterial diversity in our
experiments, we can infer the species succession based off published bacterial species
succession form SPOT (Needham and Fuhrman 2016). It has been shown that
copiotrophic bacteria, such as Flavobacteria, that are known to have higher growth rates
become abundant at SPOT during phytoplankton blooms, while slower growing bacteria,
such as SAR11 become abundant at the end of phytoplankton blooms (Needham and
124
Fuhrman 2016). Based on the observed differential bacterial SGRs in this experiment, we
hypothesize that similar bacterial species succession occurred in our experiments (Figure
4.3). Additionally, as the All treatment had the highest SPG at Tex, we hypothesize that
the availability of B
1
, B
7
, and B
12
provided a competitive advantage to groups of B-
vitamin auxotrophs. However, future studies that include bacterial diversity will be
required to determine the bacterial species composition, and the effect of B-vitamins on
determining bacterial community structure.
4.4.3 B-vitamin Mediated Bloom Succession
The observed species succession in the eukaryotic plankton community appears to
be driven by the addition of B
12
and inorganic nutrients. At T
0
eukaryotic plankton
cellular abundances were very low, less than 10 cells/ml (Figure 4.4; Table 4.3).
Dinoflagellates composed 69% of the community; Chaetoceros was the most abundant
diatom at 16%, while Ciliates accounted for 6% of the community (Figure 4.4). This
community profile was constant in the control treatment throughout the experiment, and
is consistent with what is known to be present at SPOT during its oligotrophic, fall season
(Countway et al. 2010; Hu et al. 2016; Needham and Fuhrman 2016; Schnetzer et al.
2007). At Tex, the biomass increased substantially with cellular abundances ranging
from 889 cell/ml in the nutrient treatment to 4200 cell/ml in the All treatment (Figure
4.4). The abundance of cells in the treatments containing B
12
(3500 cells/ml B
12
) were 3-
4 times higher than those containing nutrients (890 cells/ml), B
1
(1400 cells/ml), and B
7
(1200 cells/ml) alone. There was a large shift in community structure from T
0
to Tex,
where the dinoflagellete dominated community was replaced by a diatom dominated
community (Figure 4.4). Despite the difference in total cellular abundance at Tex, the
125
relative species diversity followed the same trend across all the treatments (Figure 4.4).
Chaetoceros was the dominant organism with abundances ranging from 57-80% of the
total community. Pseudonitzschia composed 8-26% of the community and dinoflagellates
made up 8-15% of the community (Figure 4.4). All other groups were less than 1% of
the total community (Figure 4.4). The shift from dinoflagelletes at T
0
to diatoms at Tex
is consistent with shifts in community driven by increased inorganic nutrient availability,
which occurs seasonally at SPOT (Countway et al. 2010; Hu et al. 2016; Needham and
Fuhrman 2016). However, the dominance of Chaetoceros, a known B
12
auxotroph, in the
B
12
treatment suggest that the increased cobalamin availability provided it with a
competitive advantage over other diatom species (Croft et al. 2006). At Tst the cellular
abundances increased an order of magnitude from Tex ranging from 2x10
4
cells/ml in the
B
1
treatment to 5.3x10
4
cells/ml in the B
7
treatment (Figure 4.4). The nutrients, B
12
, and
All treatments ranged from 3.6-3.9x10
4
cells/ml (Figure 4.4). A second shift in
community structure was observed between Tex and Tst, where the total cellular
abundance increased by an order of magnitude in all nutrient-amendment treatments,
which was driven by a bloom of Pseudonitzschia (Figure 4.4). The relative abundance of
Pseudonitzschia ranged from 42-66% of the total community (Figure 4.4). At Tst
Chaetoceros ranged from 24-46% of the community and other diatom species were
between 1-1.7% of the community (Figure 4.4). Dinoflagellates made up a smaller
portion of the community (2-6%) at Tst compared with Tex (Figure 4.4). Cylindrotheca
made up less than 1% of the community at Tex, but increased to between 2.8-6.1% of the
community at Tst (Figure 4.4).
126
Our experimental results seem to indicate that co-limitation by B-vitamins is a
driver for planktonic bloom succession at SPOT. Picoeukaryotes are co-limited by
thiamin and nutrients, and our experiment indicates they are the first species to bloom if
thiamin is present (Figure 4.1). B
12
availability appears to the driver of phytoplankton
growth, as evidenced by the higher Chl-a concentrations at Tex and the 3-4 fold higher
eukaryotic phytoplankton abundance at this point (Figure 4.4). Based on the available
literature, Chaetoceros is known to only require B
12
, and appears to be the primary driver
of this increase (Croft et al. 2006). At Tst the community shifts to Pseudonitzschia,
which is known to require B
1
, B
7
, and B
12
(Croft et al. 2006; Tang et al. 2010).
Chaetoceros appears to have a competitive advantage over Pseudonitzschia early in the
experiment in the B
12
containing treatments as Pseudonitzschia’s growth is likely
hindered by low B
1
and B
7
concentrations. As the heterotrophic bacterial community,
and Synechococcus begin to bloom after Tex, the supply of dissolved B
1
, B
7
, and B
12
likely increases, as both heterotrophic bacteria and Synechococcus are known to have B-
vitamin synthetic capabilities (Bonnet et al. 2010; Gomez-Consarnau et al. 2017; Sañudo-
Wilhelmy et al. 2014). It is likely that this secondary input of bacterioplankton produced
B-vitamins allows Pseudonitzschia to become the numerically dominate species at Tst.
Further evidence for the prokaryotic source of B-vitamins is provided by the observation
that Pseudonitzschia is most abundant at Tst in the nutrients and B
7
treatments, which
had the maximum observed Synechococcus abundances (Figure 4.1). While
Synechococcus is known to produce and utilize pseudocobalamin, and that this compound
is less bioavailable to eukaryotic phytoplankton (Heal et al. 2016; Helliwell et al. 2016),
it has been demonstrated that bacteria, which are maximally abundant in all treatments at
127
Tst, have the ability to readily remodel corrinoids, likely making pseudocobalamin
bioavailable to the phytoplankton (Gray and Escalante-Semerena 2009; Yi et al. 2012).
The eukaryotic species succession observed in this experiment, where diatom species
replaced a dinoflagellete dominated community when inorganic nutrients and B
12
were
available, either through our addition or through secondary prokaryotic cobalamin
production, is consistent with other studies where it has been shown that B
12
additions
enrich for diatoms (Koch et al. 2011). Diatoms are known to become abundant at SPOT
following the seasonal upwelling, and it has been shown that Pseudonitzschia is often the
dominant species, frequently resulting in the formation of harmful algal blooms
(Countway et al. 2010; Hu et al. 2016; Needham and Fuhrman 2016; Schnetzer et al.
2007; Venrick 2009; Venrick 2012). B-vitamins have been implicated as factors that
potentially control the onset of HABs (Gobler et al. 2007; Koch et al. 2013; Tang et al.
2010), however no studies to date have explored this hypothesis in the California Current
System or in the Southern California Bight. Pseudonitzschia was the numerically
dominate phytoplankton species at Tst in our experiment (Figure 4.4), and based off the
timing of its bloom, and its known B-vitamin auxotropy, it appears that B-vitamin
availability was a key factor in promoting its growth. Unfortunately, we lack the
taxonomic resolution and the domoic acid measurements to determine if the species of
Pseudonitzschia found in our experiment was a HAB forming species. Regardless, our
results indicate that B-vitamins influence the community composition at SPOT, and
suggest that their availability may influence the formation of HABs in the Southern
California Bight.
128
4.4.4 Particulate B-Vitamin Concentrations: Uptake v. Synthesis
Additions of dissolved thiamin (as B
1
), biotin (B
7
), and cobalamin (as CB
12
) had
varying effects on the particulate (intracellular) concentrations (pM) of these analytes and
their different biochemical forms (Figure 4.5 and Table 4.4). Initially, due to the low
biomass (0.18µg/L Chl-a), we were only able to measure the particulate concentrations of
three analytes: B
1
, B
7
, and the amino acid methionine (Met). Within the 0.2-3µm fraction
(picoplankton, PP) we observed 4.8±0.3pM B
1
, 1.2±0.4pM B
7
, and 0.1±0.07pM Met;
within the ≥3µm fraction (nanoplankton, NP) we measured 1.7±0.06pM B
1
, 2.1±0.6pM
B
7
, and 1.3±1.1 Met. All other analytes were not detectable. At T
0
B
1
, B
7
, and CB
12
were
spiked into their respective treatments so that their spike concentrations were 300pM B
1
,
100pM B
7
, and 100pM CB
12
respectively (Figure 4.5). At Tex in the B
1
addition
treatment, the mean particulate concentration of tB
1
was 914pM in the PP fraction and
1660pM in the NP fraction. The mean concentrations of tB
1
at Tst increased between 2-3
times from Tex with concentrations reaching 3162pM PP and 2135pM NP (Figure 4.5
and Table 4.4). The observed particulate concentrations of tB
1
, and B
1
(the congener
added to the treatment) are between three and ten times higher than the dissolved
concentrations added to the treatment. We calculated the expected intracellular thiamin
concentrations based on published B
1
uptake rates determined from bacterioplankton and
eukaryotic phytoplankton cultures (Koch et al. 2013). Based on these rates, uptake alone
could only account for 140pM tB
1
in the PP fraction and 84pM tB
1
in the NP fraction,
which are an order of magnitude lower than the measured intracellular tB
1
concentrations
(Figure 4.5 and Table 4.4). Therefore our data indicates that thiamin synthesis must be
occurring, and is likely the primary mechanism used by organisms from both size classes
meet their metabolic thiamin demands. At Tex the mean particulate B
7
concentrations
129
were 11pM in the PP fraction and 7.5pM in the NP fraction (Figure 4.5 and Table 4.4).
At Tst, the mean B
7
concentration increased to 16.5pM in the PP fraction and 22.8pM in
the NP fraction (Figure 4.5 and Table 4.4). The particulate B
7
concentrations were lower
than the dissolved B
7
concentration spiked into the treatment (100pM), and were lower
than the observed background dissolved concentration (57.5±10.5pM). We did not
observe any differentiated B
7
-based biological responses (e.g., biomass production and
growth) in this study (Figure 4.1, Figure 4.2, and Figure 4.3). Culture and –omics based
studies have established that the prevalence of B
7
auxotrophy in marine microbes is lower
than it is for B
1
and B
12
(Croft et al. 2006; Sañudo-Wilhelmy et al. 2014; Tang et al.
2010), potentially suggesting that the lack of B
7
-based responses was due to lower
biological demand. Mean particulate tB
12
concentrations decreased from 31.5pM at Tex
to 19.9pM at Tst in the PP fraction, but increased from 0.9pM at Tex to 1.3pM at Tst in
the NP fraction (Figure 4.5 and Table 4.4). We observed B
12
co-limitation, and it is
known that B
12
auxotrophy is the most abundant form of B-vitamin auxotrophy amongst
marine microbes, including Chaetoceros and Pseudonitzschia which were observed in
this study (Croft et al. 2006; Sañudo-Wilhelmy et al. 2014). Using published B
12
uptake
rates from bacteria and phytoplankton (Koch et al. 2013), we determined that the
expected intracellular tB
12
concentrations (50pM in the PP fraction, 2pM in the NP
fraction) are within the same order of magnitude as the observed intracellular tB
12
concentrations (31.5pM in the PP fraction, 0.9pM in the NP fraction at Tex).Therefore it
is likely that B
12
uptake was the primary B
12
acquisition strategy used by the microbial
community.
130
Treatment Date mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev.
Control T0 3/12 PP 4.77 0.303 4.77 0.303 1.23 0.381 0.108 0.0722
Control T0 3/12 NP 1.72 0.058 1.72 0.058 2.08 0.582 1.35 1.19
Control Tex 3/19 PP 76.9 82.6 56.5 79.9 20.3 2.57 0.0529 0.0748 13.0 15.4 0.268 0.379 0.225 0.318 0.0429 0.0607
Control Tex 3/19 NP 104 17.9 68.7 7.95 33.7 11.0 1.460 0.961 3.98 1.77 22.0 5.23 0.175 0.150 0.0275 0.0040 0.0246 0.0215 0.0938 0.115 0.0291 0.0176
Control Tst 3/23 PP 180 22.8 163 0.443 16.4 23.2 4.08 2.20 33.0 34.9 4.98 0.345 0.0200 0.0283 0.0123 0.0174 4.89 0.343 0.0547 0.0124
Control Tst 3/23 NP 30.2 26.4 17.2 24.4 11.4 0.0876 1.53 2.17 0.840 1.19 5.19 7.34 0.0078 0.0110 0.0072 0.0102 0.0006 0.00084
N Tex 3/19 PP 803 1140 532 752 257 363 14.7 20.8 928 980 29.9 34.7 84.0 119 4.44 6.27 0.0737 0.104 0.0908 0.128 4.25 6.01 0.0218 0.0308
N Tex 3/19 NP 539 456 403 297 130 151 6.61 7.50 30.7 42.1 3.31 1.25 244 255 0.235 0.172 0.159 0.167 0.0440 0.0058 0.0209 0.0130 0.0110 0.0027
N Tst 3/24 PP 2540 1840 1670 2360 794 562 79.9 44.5 2180 1250 68.7 30.5 1740 309 60.6 82.7 0.138 0.144 1.13 1.60 59.19 81.1 0.160 0.226
N Tst 3/24 NP 1660.00 2130 973 1190 521 732 162 218 68.1 59.7 19.1 12.1 2870 3260 1.91 2.23 0.181 0.256 1.04 1.47 0.579 0.354 0.107 0.152
B 1 Tex 3/19 PP 914 916 719 833 161 53.9 34.4 29.1 1600 920 11.5 0.0451 244 264 10.0 7.49 0.0774 0.0502 0.0848 0.0925 9.52 6.91 0.337 0.438
B 1 Tex 3/19 NP 1660 458 1220 78.2 394 339 44.3 40.0 30.6 43.2 6.55 2.50 1490 348 0.358 0.161 0.265 0.124 0.0456 0.0325 0.0341 0.0090 0.0133 0.0139
B 1 Tst 3/23 PP 3160 2310 2800 2370 355 53.4 4.55 6.43 3280 1670 17.9 7.70 1500 805 21.0 3.87 0.292 0.414 0.676 0.168 17.3 1.75 2.71 1.88
B 1 Tst 3/23 NP 2140 1220 1300 780 713 432 122 6.11 56.9 3.54 8.23 3.94 6130 6080 2.96 3.76 2.73 3.70 0.164 0.0067 0.0673 0.0483
B 7 Tex 3/19 PP 431 354 359 271 65.7 73.8 6.48 9.17 442 375 11.0 4.36 79.8 40.5 23.7 26.0 1.43 1.94 22.0 24.0 0.292 0.145
B 7 Tex 3/19 NP 664 7.59 521 13.9 118 43.6 24.7 22.1 25.8 4.48 7.56 1.18 650 36.7 0.435 0.276 0.326 0.205 0.0311 0.0175 0.0621 0.0519 0.0160 0.0021
B 7 Tst 3/24 PP 2090 1090 1440 812 545 313 104 33.7 2220 547 16.5 6.09 941 777 17.8 3.50 0.324 0.0688 1.47 1.31 14.9 1.88 1.12 0.376
B 7 Tst 3/24 NP 1300 452 775 250 439 159 81.8 42.8 56.2 45.8 22.8 0.227 2240 447 6.33 2.36 1.11 0.632 2.28 0.837 2.91 3.79 0.0238 0.0337
B 12 Tex 3/19 PP 1740 668 1380 411 350 252 11.3 4.45 2290 1720 21.5 4.83 1850 2100 31.5 15.5 0.124 0.175 0.369 0.105 30.7 15.6 0.390 0.158
B 12 Tex 3/19 NP 447 503 288 318 123 137 35.2 47.4 9.63 13.6 5.95 1.06 4330 5550 0.992 0.296 0.130 0.111 0.220 0.0852 0.537 0.0739 0.105 0.0259
B 12 Tst 3/22 PP 1990 534 1460 394 484 145 38.1 4.47 1970 383 51.4 26.6 660 217 20.0 9.38 0.323 0.223 0.476 0.0127 18.1 10.7 1.05 1.49
B 12 Tst 3/22 NP 5800 408 3940 379 1650 96.4 207 66.9 55.5 78.5 3.20 1.59 9510 10400 1.35 0.186 0.404 0.005 0.241 0.0181 0.611 0.176 0.0907 0.0135
All Tex 3/19 PP 757 9.59 535 73.8 160 19.3 61.9 83.5 670 270 15.2 7.46 353 202 14.7 4.88 0.0861 0.122 0.211 0.0776 13.0 5.46 1.47 0.383
All Tex 3/19 NP 1770 2250 1190 1480 529 694 55.5 68.7 21.2 29.9 6.47 0.902 7320 9340 2.58 2.95 1.91 2.63 0.209 0.219 0.368 0.0325 0.096 0.0639
All Tst 3/22 PP 1900 34.5 1410 429 416 293 75.3 102 2020 792 20.8 13.6 1040 676 27.7 16.4 0.811 0.321 2.39 3.18 23.1 12.2 1.42 0.734
All Tst 3/22 NP 2100 1330 1330 846 706 528 93.8 49.3 627 746 5.27 3.37 2220 1780 2.45 0.0473 1.23 0.789 0.662 0.593 0.485 0.137 0.0696 0.0115
MET CB 12 HB 12 MB 12 AB 12 tB 12 Size
Fraction
Time
Point
B 7 HMP TPP TMP B 1 tB 1
Table 4.4 Particulate B-vitamin Concentrations (pM). The means and standards deviation of the particulate B-vitamin
concentration of the biological replicates are included here. Blanks indicate that the sample was below our limits of detection.
131
Figure 4.5 Particulate B-vitamin concentrations. The concentrations of the thiamin congeners, vitamin B
7
,
and the cobalamin congeners are plotted in their respective treatments (e.g., B
1
concentrations in the B
1
addition
treatment), in order to assess B-vitamin uptake versus synthesis. The horizontal black lines on each plot
indicate the concentration of dissolved vitamin added to the treatment.
4.4.5 Estimated intracellular B-vitamin concentrations
Particulate B-vitamin concentrations were determined to be near their optimal
biochemical concentrations as defined by riboswitch Kd values. Riboswitches are
important molecular control elements where RNA interacts directly with the product
molecule that the RNA encodes to modulate the molecule’s downstream synthesis
(Madigan et al. 2015). The dissociation constant (Kd) for a riboswitch represents the
Ex St
CB
12
HB
12
AB
12
MB
12
Ex St
B
7
Ex St
B
1
TMP
TPP
0
10
20
30
0
1
2
0
5
10
15
20
0
5
10
15
20
0
1000
2000
3000
0
500
1000
1500
2000
pM
Picoplankton
Nanoplankton
+B
1
+B
7
+B
12
100pM
100pM
100pM
100pM
300pM
300pM
132
biochemically optimal intracellular concentration of the molecule; concentrations below
the Kd stimulate increased biosynthesis, while concentrations higher than the Kd repress
biosynthesis (Madigan et al. 2015). Riboswitches are known to be regulators of B-
vitamin synthesis (Mandal et al. 2003; Mandal and Breaker 2004), therefore we are able
to biochemically validate our particulate B-vitamin intracellular levels by comparing
those concentrations to the known Kd values from TPP and AB
12
riboswitches (Moore et
al. 2014; Winkler et al. 2002). Vitamins B
12
(as AB
12
) and thiamin (as TPP) measured in
the picoplankton size fraction (0.2-3µm) were used for these calculations as the known B-
vitamin riboswitch Kd values have been determined in bacteria (Moore et al. 2014;
Winkler et al. 2002). Total cellular abundance was calculated by summing the observed
abundances of heterotrophic bacteria, Synechococcus, and picoeukaryotes. These three
measured classes of organisms span all three domains of life, and have considerable
variations in cellular size equating to a range of possible biovolumes spanning four orders
of magnitude from 4.2x10
-3
µm
3
for bacteria to14µm
3
for picoeukaryotes (Caron et al.
2017; Pomeroy et al. 2007; Waterbury et al. 1986). A midpoint biovolume (cellular
diameter 1µm, 1.77µm
3
) was used to normalize the particulate B-vitamin concentrations.
All measurements from all treatments and time-points for each vitamin were binned. The
resulting estimated intracellular concentration (moles B-vitamin per liter biovolume) for
TPP had an IQR of 3.89x10
-7
-7.42x10
-6
M (Figure 4.6), and the established TPP
riboswitch Kd value, 6.0x10
-7
M (Winkler et al. 2002), falls within the lower end of the
IQR. AB
12
has an estimated intracellular concentration range IQR of 7.15x10
-9
- 3.18x10
-
8
M (Figure 4.6). The AB
12
riboswitch Kd value is 5x10
-9
M (Moore et al. 2014), which
falls just below the observed AB
12
IQR. The fact that both of these established
133
riboswitch values falls within the observed concentration ranges of their respective
molecules, despite the diversity found in our environmental samples, suggests that the
concentrations reported here are biochemically relevant. In order to gain a broader
prospective on the biochemical relevance of our data, the estimated intracellular
concentration of the amino acid methionine was also estimated, resulting in an IQR of
1.47x10
-5
-9.47x10
-5
M (Figure 4.6). This range was compared to established total
intracellular free amino acid concentrations which are known to be within the hundreds
of micro moles per liter biovolume (Groen et al. 1982; Hochachka and Somero 2002).
The observed IQR of methionine was an order of magnitude lower than the total amino
acid pool (20 amino acids; Figure 4.6). Additionally, the ranges of intracellular
concentrations for TPP and AB
12
were between two and four orders of magnitude lower
than the intracellular concentrations of the total amino acid pool, which is consistent with
the biological demand for amino acids versus coenzymes.
Figure 4.6 Estimated intracellular B-vitamin concentrations are near biochemically optimal B-vitamin
riboswitch Kd values. Intracellular concentrations (M, moles B-vitamin per liter biovolume) of TPP, AB12, and
methionine were estimated using the observed particulate B-vitamin concentrations from the 0.2-3 µm size
fraction normalized using the total cellular abundance and a midpoint biovolume estimate (1.77µm
3
).
Observations from all treatments and timepoints were binned into the boxplots shown above. The red lines are
riboswitch Kd values, and the orange line is the total free amino acid concentration.
0e+00
3e−08
6e−08
9e−08
0e+00
2e−04
4e−04
0.00e+00
2.50e−06
5.00e−06
7.50e−06
1.00e−05
1.25e−05
Intracellular Concentration (M)
TPP Methionine AB
12
134
4.4.6 B-Vitamin Quotas within Specific Experimental Treatments
Particulate B-vitamin concentrations were normalized to the observed
concentrations of particulate organic nitrogen (PON) in order to correct for the
differential biomass found in each biological replicate (Menden-Deuer and Lessard
2000), and to calculate a bulk community B-vitamin quota. Despite high technical
precision during our B-vitamin analysis, there was substantial variability of B-vitamin
quotas between biological replicates (Table 4.5). Within the PP fraction of the B
1
treatment, the mean tB
1
quota (pmole vitamin/µmole PON) was 110 at Tex and 165 at Tst
(Figure 4.7 and Table 4.5). The B
1
quotas are the largest contributor to the tB1 quota in
both fractions (Figure 4.7). The mean B
1
quotas in the PP fraction at Tex and Tst were 85
and 146 B
1
, 21 and 18 TMP, and 4.2 and 0.2 TPP respectively (Figure 4.7). In the NP
fraction the mean tB
1
quota dropped from 221 at Tex to 112 at Tst (Figure 4.7). The
mean thiamin normalized congener quotas for B
1
and TMP decreased in the NP fraction
between Tex and Tst (from 168 to 68 B
1
and 49 to 37 TMP), while the mean quota for
TPP increased from 5.4 to 6.5 (Figure 4.7). The mean tB
1
quotas from both size fractions
ranged from 89 to 283 pmole vitamin/µmole PON, which is within the tB
1
inter-quartile
range (IQR) previously reported in microbial communities from the Mediterranean Sea
(Suffridge et al. 2017b). Additionally, the relative fractionation of the thiamin congeners
within the tB
1
pool is similar to what has been observed in other bulk environmental B-
vitamin quotas, where the B
1
quota is highest and the TPP quota is the lowest (Suffridge
et al. 2017b) . The magnitude of the tB
1
quotas suggests that growth rate is controlling
the metabolic demand for thiamin, as the highest tB
1
quota in the NP fraction was
observed at Tex when the chlorophyll based growth rates were the highest (Figure 4.3).
Evidence for thiamin synthesis can potentially be found by observing the magnitude of
135
the quotas of the thiamin precursor moiety, HMP. The mean HMP quotas in the B
1
treatment decreased from Tex to Tst in both PP and NP fractions; 206-173 pmole
vitamin/µmole PON in the PP fraction and 4.9-3.0 pmole vitamin/µmole PON in the NP
fraction (Figure 4.7). The quota values for the PP fraction fall within the previously
reported HMP quota IQR of 11-104 pmole vitamin/µmole PON (Suffridge et al. 2017b).
HMP is used as a precursor moiety in both the prokaryotic and eukaryotic thiamin
biosynthesis (Begley 1996; Begley et al. 1999; Chatterjee et al. 2006; Edwards et al.
2017; Kraft and Angert 2017). The two order of magnitude difference in the mean HMP
quota values between the PP and NP fractions (Figure 4.7), could be indicative of higher
rate of thiamin biosynthesis occurring within the PP fraction at this point where maximal
SGRs for heterotrophic bacteria were observed (Figure 4.3). Thiamin is an essential
coenzyme for central metabolic processes including both respiration and carbon fixation
via the TCA and Calvin cycles respectively (Begley 1996; Begley et al. 1999; Berg et al.
2007), therefore its cellular demand, and its rates of synthesis, is likely highest when rates
of metabolic activity are highest.
136
Figure 4.7 Vitamin B
1
congener and HMP quotas (pmole vitamin/µmole PON) in the B
1
treatment. The
mean quota values for each analyte are presented.
0
50
100
150
0
50
100
150
200
Ex St
pmole pvit/μM PON
B
1
TMP
TPP
0
50
100
150
200
0
1
2
3
4
5
Ex St
HMP
Picoplankton Nanoplankton
+B
1
Treatment
137
Treatment Date mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev. mean st.dev.
Control T0 3/12 PP 12.3 0.782 12.3 0.782 3.18 0.986 0.279 0.187
Control T0 3/12 NP 4.44 0.149 4.44 0.149 5.37 1.50 3.48 3.09
Control Tex 3/19 PP 45.3 48.5 33.2 47.0 12.0 1.41 0.0311 0.0440 7.74 9.18 0.158 0.223 0.132 0.187 0.0253 0.0357
Control Tex 3/19 NP 61.6 11.2 40.7 5.06 20.0 6.66 0.862 0.561 2.35 1.02 13.0 3.21 0.103 0.0880 0.0163 0.00250 0.0145 0.0126 0.0552 0.0676 0.0172 0.0103
Control Tst 3/23 PP 105 13.6 95.0 0.0344 9.57 13.5 2.38 1.29 19.1 20.2 2.89 0.209 0.0116 0.0164 0.00717 0.0101 2.84 0.208 0.0318 0.00729
Control Tst 3/23 NP 17.6 15.4 10.0 14.2 6.64 0.0306 0.893 1.26 0.49 0.692 3.02 4.28 0.00453 0.0064 0.00418 0.00592 0.00035 0.0005
N Tex 3/19 PP 153 217 101 143 48.9 69.2 2.80 3.96 172 194 4.47 4.87 16.0 22.7 0.846 1.20 0.0140 0.0199 0.0173 0.0245 0.810 1.15 0.00415 0.00587
N Tex 3/19 NP 97.9 93.9 72.4 62.9 24.3 29.6 1.23 1.47 5.84 8.05 0.576 0.317 45.1 50.6 0.0368 0.0213 0.0241 0.0230 0.00749 0.00238 0.00330 0.00151 0.00189 0.000809
N Tst 3/24 PP 136 102 90.1 127 41.2 27.9 4.24 2.50 116 70.1 3.65 1.74 91.9 19.8 3.27 4.47 0.00713 0.00730 0.0609 0.0862 3.19 4.38 0.00864 0.0122
N Tst 3/24 NP 89.3 116 52.4 64.3 28.1 39.6 8.77 11.8 3.64 3.28 1.01 0.675 154 177 0.102 0.121 0.00976 0.0138 0.0561 0.0794 0.0308 0.0198 0.00579 0.00819
B 1 Tex 3/19 PP 110 94.7 84.9 90.1 21.4 1.80 4.25 2.82 206 70.9 1.60 0.411 29.1 28.0 1.26 0.685 0.00983 0.00424 0.0101 0.00982 1.20 0.622 0.0389 0.0489
B 1 Tex 3/19 NP 222 5.02 168 32.1 48.6 33.2 5.43 3.98 4.99 7.06 0.951 0.576 213 101 0.0467 0.0097 0.0345 0.00783 0.00575 0.00290 0.00487 0.00244 0.00159 0.00145
B 1 Tst 3/23 PP 166 115 146 119 19.0 3.77 0.251 0.355 173 80.3 0.946 0.364 78.9 39.0 1.12 0.261 0.0161 0.0228 0.0359 0.00722 0.926 0.138 0.147 0.107
B 1 Tst 3/23 NP 112 59.4 68.4 38.2 37.4 21.2 6.47 0.0110 3.03 0.0414 0.444 0.231 319 308 0.163 0.208 0.150 0.204 0.00876 0.000780 0.00365 0.00275
B 7 Tex 3/19 PP 65.3 53.6 54.3 41.0 9.95 11.2 0.982 1.39 67.0 56.8 1.67 0.663 12.1 6.15 3.60 3.95 0.217 0.294 3.34 3.64 0.0443 0.0220
B 7 Tex 3/19 NP 101 1.28 79.0 2.01 17.9 6.64 3.74 3.35 3.92 0.684 1.15 0.177 98.5 5.44 0.0660 0.0420 0.0494 0.0311 0.00472 0.00266 0.00942 0.00788 0.00242 0.00033
B 7 Tst 3/24 PP 108 50.2 74.2 37.7 28.1 14.6 5.52 2.13 116 21.1 0.859 0.264 48.2 37.7 0.945 0.246 0.0169 0.00250 0.0797 0.0741 0.789 0.151 0.0597 0.0237
B 7 Tst 3/24 NP 69 28.3 41.2 15.9 23.4 9.89 4.38 2.53 2.88 2.22 1.20 0.0911 117 15.8 0.329 0.102 0.0595 0.0371 0.122 0.0520 0.147 0.190 0.00119 0.00169
B 12 Tex 3/19 PP 132 10.8 106 0.936 25.1 11.7 0.856 0.0800 164 82.2 1.79 0.920 124 123 2.35 0.469 0.00784 0.0111 0.0310 0.0176 2.28 0.498 0.0335 0.0224
B 12 Tex 3/19 NP 29.8 29.5 19.3 18.6 8.26 8.01 2.26 2.96 0.608 0.861 0.493 0.232 281 340 0.0763 0.0006 0.00911 0.00576 0.0167 0.00145 0.0424 0.00733 0.00813 0.000496
B 12 Tst 3/22 PP 92.2 20.3 68.0 15.0 22.4 5.66 1.78 0.297 91.8 13.3 2.43 1.36 30.6 8.58 0.944 0.485 0.0153 0.0112 0.0222 0.00051 0.859 0.540 0.0475 0.0671
B 12 Tst 3/22 NP 271 32.5 185 26.8 77.1 8.33 9.60 2.65 2.68 3.80 0.148 0.0668 432 465 0.0627 0.0056 0.0189 0.00068 0.0112 0.0003 0.0284 0.00682 0.0043 0.000842
All Tex 3/19 PP 57.0 2.83 40.4 7.06 12.1 1.90 4.55 6.13 50.2 18.5 1.16 0.605 26.3 14.2 1.10 0.327 0.00666 0.00942 0.0160 0.00644 0.970 0.376 0.112 0.0330
All Tex 3/19 NP 137 174 91.5 115 40.9 53.8 4.28 5.33 1.64 2.32 0.489 0.0860 565 724 0.198 0.229 0.147 0.204 0.0161 0.0171 0.0278 0.00347 0.00731 0.00508
All Tst 3/22 PP 92.0 2.99 68.3 21.8 20.1 13.9 3.62 4.88 97.9 37.0 1.02 0.674 50.4 32.0 1.35 0.816 0.0395 0.0161 0.117 0.156 1.12 0.607 0.0692 0.0366
All Tst 3/22 NP 102 65.7 63.2 41.9 34.4 26.1 4.53 2.33 30.1 35.8 0.257 0.167 107 84.9 0.119 0.0006 0.0594 0.0374 0.0323 0.0292 0.0236 0.00699 0.00338 0.00061
TPP
Time Point
Size
Fraction
tB 1 B 1 TMP HB 12 CB 12 HMP B 7 MET tB 12 AB 12 MB 12
Table 4.5 Particulate B-vitamin Quotas (pmole vitamin/µmole PON). The biological replicates were individually
normalized with their respective PON concentrations. The means and standard deviations of the PON normalized biological
replicates are included here. Blanks indicate that the sample was below our limits of detection.
138
Figure 4.8 Vitamin B
7
quotas (pmole vitamin/µmole PON) in the B
7
treatment. The mean quota values for
each analyte are presented.
In the B
7
treatments, the differential responses of B
7
quotas in each size fraction
seem to indicate that organisms within the PP fraction have growth rate dependent B
7
0.0
0.5
1.0
1.5
0.00
0.25
0.50
0.75
1.00
1.25
Ex St
pmole pvit/μM PON
B
7
+B
7
Treatment
139
requirements, whereas organisms within the NP fraction have a constant B
7
requirement.
These observations are of particular interest as no B
7
-based differentiation in biomass
standing stocks or growth rates were observed (Figure 4.1, Figure 4.2, Figure 4.3, and
Figure 4.4). Within the PP fraction, mean vitamin B
7
quotas decreased two fold from
1.7pmole vitamin/µmole at Tex to 0.85pmole vitamin/µmole at Tst (Figure 4.8). Within
the NP fraction the quotas remained essentially constant between the two time points
varying between 1.1 pmole vitamin/µmole at Tex and 1.2 pmole vitamin/µmole at Tst
(Figure 4.8). The B
7
quotas observed in this experiment were between 6-10 times lower
than the previously reported 6.3-23 pmole vitamin/µmole PON IQR of B
7
quotas, and the
dissolved B
7
concentration were an order of magnitude lower than the 0.5nM mean
dissolved B
7
concentration found in the Mediterranean Sea (Suffridge et al. 2017b). The
known requirements for exogenous vitamin B
7
in marine microbes are known to be lower
than those for B
1
and B
12
(Sañudo-Wilhelmy et al. 2014). Based on published uptake
rates from phytoplankton (Tang et al. 2010), we calculated that the median expected
intracellular quota for B
7
is 0.15 pmole vitamin/µmole PON. Our observed B
7
quotas are
an order of magnitude greater than the uptake-estimated value, suggesting that B
7
synthesis at SPOT within both fractions outpaces uptake (Figure 4.8).
140
Figure 4.9 Vitamin B
12
congener and methionine quotas (pmole vitamin/µmole PON) in the B
12
treatment.
The mean quota values for each analyte are presented.
Within the B
12
treatment, the mean tB
12
quotas in both PP and NP fractions are
highest when the growth rate is highest (at Tex), however the magnitude of the quota and
partitioning of the cobalamin congeners show clear differences between the size fractions
0.0
0.5
1.0
1.5
2.0
0.00
0.02
0.04
0.06
0.08
Ex St
pmole pvit/μM PON
CB
12
HB
12
AB
12
MB
12
0
40
80
120
0
100
200
300
400
Ex St
MET
+B
12
Treatment
141
(Figure 4.9). Within both the PP and NP fractions the mean tB
12
quotas dropped from
Tex to Tst (from 2.3-0.9 pmole vitamin/µmole PON in the PP and 0.08-0.06 pmole
vitamin/µmole PON in the NP; Figure 4.9). The observed SGR for heterotrophic bacteria
and phytoplankton (Chl-a based) were highest at Tex, providing evidence that the
increased growth rate increases cellular B
12
requirement. The tB
12
quotas observed here
are at most three times lower than the IQR tB
12
quotas (7.1-32 pmole vitamin/µmole
PON) measured in field microbial communities in the Mediterranean Sea (Suffridge et al.
2017b). The highest quotas in the Mediterranean Sea were found in oligotrophic regions,
while lower quotas were found in the regions with highest bacterial activity (Suffridge et
al. 2017b). While we did not measure bacterial activity, the SGRs that we calculated
indicate that the community in our experiment was actively growing, and therefore, based
off the environmental observations in the Mediterranean Sea, it can be expected to have
lower cobalamin cellular quotas. The relative abundance of cobalamin congeners with
respect to the tB
12
pool in the PP fraction are similar to those previously reported, with
HB
12
making up the majority of the tB
12
pool (Figure 4.9; Suffridge et al. 2017b). In the
PP fraction, between Tex and Tst, the mean HB
12
quota dropped from 2.3-0.8 pmole
vitamin/µmole PON, the mean AB
12
quota remained constant at 0.01pmole
vitamin/µmole PON, and the CB
12
and MB
12
quotas only varied slightly: 0.03-0.05 pmole
CB
12
/µmole PON, 0.03-0.02 pmole MB
12
/µmole PON (Figure 4.9). The relative
abundances of the cobalamin congeners in the NP fraction are remarkably different from
the PP fraction, with the active forms of B
12
(AB
12
and MB
12
) accounting for about half
of the tB
12
pool (Figure 4.9). The differences in congener relative partitioning and the
magnitude of the tB
12
suggest that the organisms present in the PP and NP fractions have
142
separate cobalamin requirements and cobalamin related metabolic strategies.
Additionally, cobalamin synthesis is absent from the NP fraction as the cobalamin
synthesis pathway is only present in prokaryotes (PP fraction) (Banerjee 1997; Banerjee
and Ragsdale 2003; Brown 2005). Therefore, while de-novo cobalamin synthesis is
potentially enriching the tB
12
quotas in the PP fraction, the quotas in the NP fraction
potentially represent the metabolic demand for cobalamin in the absence of enrichment
via biosynthesis. CB
12
was the form of cobalamin added to the experiment, and the CB
12
quota was relatively more abundant in the NP fraction than the PP fraction (Figure 4.9).
The cells in the NP fraction are likely assimilating the available CB
12
, and remodeling it
to the active forms to meet their cellular needs. As many eukaryotes obligately require
cobalamin, yet none can synthesize it, they are likely evolved to keep cellular quotas of
cobalamin to a minimum while maximizing the coenzyme’s catalytic potential. In
contrast, bacteria contain both cobalamin synthesizers and auxotrophs, and it is well
known that even those organisms with B-vitamin synthesizing capabilities will take
exogenous B
12
if its available in the environment (Bonnet et al. 2010; Evans et al. 2015).
The primary catalytic role of cobalamin is the synthesis of the amino acid
methionine (via the methionine synthase gene MetH), as a result the quotas of these two
organic molecules have been observed to be highly correlated (Suffridge et al. 2017b).
Methionine quotas were observed to have large fluctuations between time points during
our amendment experiments (Figure 4.9). In the PP fraction the mean quota dropped
from 123 pmole vitamin/µmole PON at Tex to 30.6 pmole vitamin/µmole PON at Tst
(Figure 4.9). The opposite trend was observed in the NP fraction where the mean quota
increased from 281 at Tex to 431 at Tst (Figure 4.9). The values at Tst in NP fraction fall
143
just above the previously reported IQR for methionine (4.1-25 pmole vitamin/µmole
PON) measured in the Mediterranean Sea (Suffridge et al. 2017b). However in both size
fractions at Tex and in the PP fraction at Tst, the methionine quotas are an order of
magnitude greater than what has been previously reported in the Mediterranean Sea
(Suffridge et al. 2017b) . The increased growth rates in the B
12
treatment, is likely
responsible for these elevated methionine quotas. Increased growth rates require an
increased rate of protein synthesis (Madigan et al. 2015), and hence an increased
methionine quota. Both tB
12
and methionine quotas are elevated at this point, providing
direct evidence for cobalamin dependent methionine synthesis. MB
12
is the active form
of cobalamin involved in methionine synthesis, where its methyl group is transferred to
cysteine, synthesizing methionine (Berg et al. 2007). After the catalytic event, MB
12
becomes HB
12
and MB
12
is ultimately regenerated (Koutmos et al. 2009). Therefore, the
high HB
12
intracellular quotas could be indicative of high rates of catalytic MB
12
turnover
resulting in high methionine quotas (Figure 4.9). The methionine quotas in the NP
fraction do not appear to be directly linked to growth rate, as the highest quota was
observed at Tst when Chl-a based SGR was the lowest (Figure 4.3 and Figure 4.9).
Within the NP fraction we observed a clear shift in the eukaryotic plankton community
from Tex where Chaetoceros was dominant to Tst where Pseudonitzschia was dominant
(Figure 4.4). It is possible that this shift in community is responsible for the increase in
methionine quota at Tst, as Pseudonitzschia might have higher methionine requirements
than Chaetoceros, although future studies will be required to determine the methionine
requirements of these diatoms.
144
4.4.7 B-vitamin Synergy: B
12
quotas in all treatments
Cobalamin is the most commonly required B-vitamin in marine microbial
communities (Croft et al. 2005; Croft et al. 2006; Droop 1957; Gobler et al. 2007; Koch
et al. 2012; Koch et al. 2011; Koch et al. 2013; Sañudo-Wilhelmy et al. 2014; Tang et al.
2010), and we observed B
12
co-limitation in this study; therefore, in order to better
understand microbial cobalamin requirements, we determined the B
12
and methionine
quotas in all treatments (nutrients, B
1
, B
7
, B
12
, and All amendments). The addition of B
12
did not appear to have a differential impact on the tB
12
cellular quotas within both size
fractions (Figure 4.10 and Table 4.5), which potentially suggests that cobalamin
uptake/synthesis is tightly regulated within the domains of life (e.g., bacteria and archaea
v. eukaryotes). The PP fraction contains all known organisms with cobalamin synthesis
capacity (Banerjee and Ragsdale 2003; Sañudo-Wilhelmy et al. 2014). Across all
treatments, this fraction contained the highest tB
12
quotas ranging from 0.84 to 3.6pmole
vitamin/µmole PON (Figure 4.10). HB
12
was the dominate congener in all treatments
with relative abundance of the tB
12
pool ranging from 83-98% (Figure 4.10), suggesting
that de novo synthesis by prokaryotes, or rapid usage and slow regeneration to MB
12
was
occurring (Brown 2005; Koutmos et al. 2009). The tB
12
quotas in the NP fraction were an
order of magnitude lower than in the PP fraction with quotas ranging from 0.06 to
0.16pmole vitamin/µmole PON (Figure 4.10). The NP fraction exclusively contains
organisms that are unable to synthesize cobalamin (Banerjee and Ragsdale 2003; Sañudo-
Wilhelmy et al. 2014), therefore it is possible that these lower quota values are
representative of actual cellular metabolic demand for cobalamin as opposed to the PP
fraction where poorly regulated cobalamin synthesis (even in just a few groups of
organisms) could be responsible for increasing the observed quotas. The active forms of
145
cobalamin (AB
12
and MB
12
) were much more abundant in the NP fraction where the
median relative abundance of the active forms across all treatments was 58% for AB
12
and 19% for MB
12
, while the inactive forms medians were 4% CB
12
and 17% HB
12
(Figure 4.10). AB
12
had the highest median relative abundance in the NP fraction,
despite the fact that MB
12
is known to be the active congener with highest cellular
requirements (Brown 2005; Matthews et al. 2003). The highest AB
12
quotas were
observed in both B
1
containing treatments (B
1
, and All), potentially suggesting a link
between a B
1
and B
12
catalyzed processes (Figure 4.10). Our data indicate that thiamin
synthesis was occurring in the B
1
addition treatment, and potential link can be found in
the thiamin synthesis pathway, where several radical mediated rearrangements of the
ribonucleotide precursor to HMP are required (Chatterjee et al. 2008). While the role of
AB
12
in this process has not been defined, this vitamin is known to catalyze radical based
reactions, including the transfer of adenosyl groups, similar to those required for HMP
synthesis (Brown 2005), therefore it is conceivable that it could also be involved in this
process. While MB
12
was not the most abundant chemical form of the tB
12
, its role as a
coenzyme in cobalamin dependent methionine synthase is well defined (Matthews et al.
2003); Therefore if methionine quotas are used as a proxy for MB
12
catalytic activity
(Banerjee and Matthews 1990; Matthews et al. 2003), the highest MB
12
activity is found
in the B
12
addition in the NP size fraction as this treatments have the roughly double the
methionine quotas as the no vitamin addition treatments (Table 4.5). Therefore, even
though the tB
12
quotas are lower in the NP fraction, and the tB
12
quotas in the B
12
addition of the NP fraction are not the highest, this fraction does appear to have the
highest rate of B
12
mediated catalytic activity. Additionally, the highest CB
12
quota
146
values were observed within the B
12
addition (11% Tex, 7% Tst), suggesting that when
exogenous B
12
is supplied (CB
12
was the form that was added), it will be utilized,
remodeled, and utilized for cobalamin dependent methionine synthesis (Figure 4.10).
147
Figure 4.10 Synergistic effects of B-vitamin addition treatments on B
12
congener quotas (pmole
vitamin/µmole PON). The mean B
12
congener quota values in each experimental treatment are shown.
Conclusion 4.5
In this study we explored the impact of B-vitamin additions on microbial species
succession in a coastal upwelling system in the Southern California Bight at the San
Pedro Ocean Time series (SPOT). The oceanographic conditions at SPOT were
uncharacteristically oligotrophic during our grow-out experiments in March 2015 due to
the 2015 El Niño. We determined that phytoplankton species were co-limited by nutrients
and B
12
in this experiment. The chain forming diatom Chaetoceros, a known B
12
0.0
0.4
0.8
1.2
0
1
2
3
0.0
0.5
1.0
1.5
2.0
0.0
0.5
1.0
0.00
0.05
0.10
0.15
0.0
0.1
0.2
0.3
0.00
0.02
0.04
0.06
0.08
0.00
0.05
0.10
0.15
0.20
Ex St Ex St Ex St Ex St
pmole pvit/μM PON
Picoplankton Nanoplankton
CB
12
HB
12
AB
12
MB
12
+N
+B
1
+B
7
+B
12
+All
0
1
2
3
0.000
0.025
0.050
0.075
0.100
Ex St
148
auxotroph, was initially dominant in the B
12
treatment at the exponential time point,
which suggests that increased cobalamin availability provided it with a competitive
advantage over other diatom species. Pseudonitzschia, a pennate diatom known to
produce HABs, became the numerically dominant species at stationary phase in all
treatments. This diatom is a known vitamin B
1
, B
7
, and B
12
auxotroph; therefore its late
bloom is likely due to secondary input from bacterioplankton produced B-vitamins. The
particulate B-vitamin concentrations were measured in each treatment and size fraction,
and were compared to quotas estimated from B-vitamin uptake rate experiments. Those
results indicate that B
1
synthesis and B
12
uptake were the dominant processes that
organisms used to satisfy their B-vitamin requirements. Our results indicate that
cobalamin remodeling is occurring, as CB
12
(the form of cobalamin added to the
experiment) was the least abundant B
12
congener in both size fractions (11% NP v 1%
PP), while HB
12
was the most abundant. The observed tB
1
quotas (pmole vitamin/µmole
PON) were determined to be growth rate dependent, with quotas in the NP fraction being
two times higher at Tex than Tst, and within the PP fraction the TPP quota being 16 times
higher at Tex than during Tst. The tB
12
quotas are also growth rate dependent, and were
2.5 times higher in the PP fraction and 1.2 times higher in the NP at Tex than at Tst. The
B
12
quotas are an order of magnitude higher in the PP fraction, suggesting that the lack of
the cobalamin biosynthesis pathway in eukaryotes has reduced their cellular requirements
for this coenzyme. For the first time, we have provided evidence that growth rate
influences B-vitamin quotas in marine microbes, suggesting that dose-response method
of estimating B-vitamin requirements may underestimate B-vitamin requirements by as
much as an order of magnitude by not measuring the effect of growth rate. Additionally,
149
our findings indicate that exogenous B-vitamin availability causes shifts in phytoplankton
community structure, which has the potential to influence the strength of the biological
carbon pump.
Acknowledgements 4.6
We thank Lynda Cutter, Erin McParland, Nancy Tenenbaum, Naomi Levine, Troy
Gunderson, Danielle Monteverde, Babak Hassanzadeh, and the Hutchins Lab for their
help and support with environmental sample collection, experimental setup, sample
processing, and manuscript preparation. Additionally, we thank the captain and crew of
the R/V Yellowfin for their assistance in sample collection. Financial support for this
research was provided by NSF Biological Oceanography grant number OCE 1435666.
150
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Chapter 5 Conclusions and Future Directions
This dissertation examines the ways that multiple biochemically relevant B-vitamins
and their congeners impact marine microbial ecology. This work contains the first ever
measurements of B-vitamin congeners in the particulate and dissolved fractions in the
ocean. These novel paired measurements enable marine microbial ecology to be
examined from a new prospective, which allows for a greater understanding of the factors
controlling microbial growth, diversity, and community structure.
The second chapter of this dissertation describes the development a direct method for
the determination of particulate and dissolved B-vitamins in the ocean. The particulate
protocol involves a cellular extraction from suspended particles, followed by a liquid
phase extraction to remove hydrophobic molecules that could interfere with the analysis.
The dissolved method uses a C
18
resin to preconcentrate B-vitamins from seawater.
Analysis for both vitamins pools is carried out using a gradient elution using a C
18
column, followed by direct quantification using a LC/MS. This new analytical protocol is
used to quantify multiple biochemical congeners of thiamin and cobalamin including B
1
,
TMP, TPP, AB
12
, CB
12
, MB
12
, and HB
12
. Additionally, the thiamin precursor HMP,
vitamin B
7
, and the amino acid methionine are also measured. This method is sensitive
and accurate, and enables simultaneous direct quantification of particulate and dissolved
B-vitamins in the ocean.
The third chapter of the dissertation presents the most complete oceanographic B-
vitamin dataset assembled to date, including paired particulate and dissolved
measurements of multiple biochemical B-vitamin congeners. We use these data to
158
establish spatial distributions of these essential coenzymes spanning distinct
microbiological and oceanographic regimes in the Mediterranean Sea and the Atlantic
Ocean. All B-vitamin congeners were present in both the particulate and the dissolved
pools across the transect, however, as has previously been reported, large B-vitamin
depleted regions were observed. Dissolved B-vitamins co-occurred in geographically
distinct patches where the concentrations were an order of magnitude higher than the
areas surrounded the geographical region. The dissolved B-vitamin-enriched areas
occurred in distinct water masses characterized by increases in biological productivity.
Additionally, these vitamin-enriched areas were generally found in zones with slightly
negative N* values, suggesting that phosphorus limitation was alleviated. The relative
abundances of B-vitamin congeners indicated a decoupling between the biochemical
intracellular pool and the exogenous dissolved fraction, as MB
12
was the most abundant
cobalamin congener in the dissolved pool (IQR 52-70%) while HB
12
was most abundant
in the intracellular fraction (IQR 62-86%). B
1
was the most abundant congener in both
the dissolved and particulate pools (IQR 71-83% dissolved 47-59% particulate), however
the magnitude of this abundance was about 1.5 times greater in the dissolved pool.
Additionally, particulate B-vitamin quotas of the bulk microbial community were
calculated. These intracellular quotas are the first directly measured estimates of
environmental B-vitamin biological requirements in marine systems. Maximum quotas
were measured at DCM depths where the values were 2-10 times higher than the median
quota value, suggesting that increased rates of biological activity increase B-vitamin
requirements. Biogeographic trends in median quota values were also observed. For
example, SAR11 clade of bacteria is known to require HMP, and the median HMP quotas
159
were between 4-7x higher in the regions with the greatest LNA (SAR11) abundances,
suggesting that the differentiation in B-vitamin quota values is caused by differential
microbial B-vitamin requirements. Finally, we used linear regression models to link the
abundances of microbes with B-vitamin auxotrophy to the exogenous availability of the
compounds that they require. We found that although the non-active forms (CB
12
and
HB
12
) were the least abundant dissolved congeners, they were the congeners most
commonly correlated with microbial abundance, suggesting that they are the most labile
forms of cobalamin. Vitamin B
7
was correlated in all but one of the significant models
(heterotrophic bacteria in the western region of the Mediterranean), which suggest that B
7
might be a more important player in microbial growth than previously thought.
Methionine was also correlated in 70% of the significant models, which is consistent with
the known uptake of amino acids from the dissolved pool, however the oceanic
distribution of this amino acid is highly understudied and warrants more attention.
Finally, the fourth chapter explores the impact of B-vitamin additions on microbial
species succession in a coastal upwelling system in the Southern California Bight at the
San Pedro Ocean Time series (SPOT). The oceanographic conditions at SPOT were
uncharacteristically oligotrophic during our study in March 2015 due to the El Niño
conditions. We determined that phytoplankton were co-limited by nutrients and B
12
in
this experiment. The chain forming diatom Chaetoceros, a known B
12
auxotroph, was
initially dominant in the B
12
treatment at the exponential time point, which suggests that
the increased cobalamin availability provided it with a competitive advantage over other
diatom species. Pseudonitzschia, a pennate diatom known to produce HABs, became the
numerically dominant species at stationary phase in all treatments. This diatom is a
160
known vitamin B
1
, B
7
, and B
12
auxotroph; therefore its late bloom is likely due to
secondary input of bacterioplankton produced B-vitamins. The particulate B-vitamin
concentrations were measured in each treatment and size fraction, and were compared to
quotas estimated from B-vitamin uptake rate experiments, and they indicate that B
1
synthesis and B
12
uptake were the principal B-vitamin acquisition processes. Our results
indicate that cobalamin remodeling is occurring, as CB
12
(the form of cobalamin added to
the experiment) was the least abundant B
12
congener in both size fractions (11% NP v 1%
PP). The observed tB
1
quotas (pmole vitamin/µmole PON) were determined to be growth
rate dependent, with quotas in the NP fraction being two times higher at Tex than Tst,
and within the PP fraction the TPP quota being 16 times higher at Tex than Tst. The tB
12
quotas are also growth rate dependent, and were 2.5 times higher in the PP fraction and
1.2 times higher in the NP at Tex than at Tst. The B
12
quotas are an order of magnitude
higher in the PP fraction, suggesting that the lack of the cobalamin biosynthesis pathway
in eukaryotes has reduced their cellular requirements for this coenzyme. For the first
time, we have provided evidence that growth rate influences B-vitamin quotas in marine
microbes, suggesting that dose-response method of estimating B-vitamin requirements
may underestimate B-vitamin requirements by as much as an order of magnitude by not
measuring the effects of growth rate. Additionally, our findings indicate that exogenous
B-vitamin availability causes shifts in phytoplankton community structure, which has the
potential to influence the strength of the biological carbon pump.
Research on B-vitamins in the marine environment has provided many testable
hypotheses. For example, how does B-vitamin availability and B-vitamin mediated
microbial species succession impact the control of the biological carbon pump in the
161
open ocean? Does exogenous B-vitamin availability affect N
2
fixation, and are
diazotrophs sources or sinks of B-vitamins in the community? What are the B-vitamin
quotas for individual species of bacteria and eukaryotes? What are the rates of B-vitamin
synthesis in specific species? What factors influence these rates? What are the rates of B-
vitamin release (excretion, lysis, etc.) into the dissolved pool? What is the stability of B-
vitamins in seawater? What are their residence times in the dissolved pool?
Research creates more questions than it answers, and the investigations of B-vitamin
in the marine environment are no exception. This is an exciting new area of study, and
the work that I have done in the course of this dissertation has created many exciting new
unexplored hypotheses. It is my hope that the data presented in this dissertation will help
future researchers advance our understanding of the role B-vitamins play in the ocean.
Abstract (if available)
Abstract
Phytoplankton are major components of the global environment and are responsible for about half of the global primary production (Field et al. 1998). They support the base of the oceanic food web, and mediate carbon flux from the atmosphere to the deep ocean via the biological carbon pump (Chisholm 2000). Phytoplankton are of such importance to the global environment that much research has been conducted to elucidate the factors that control their growth and community structure. For example, during the past several decades, biogeochemical studies have focused primarily on the role that inorganic macronutrients and some trace metals play on regulating phytoplankton dynamics in the world ocean (Boyd et al. 2000
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Suffridge, Christopher Philip
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Core Title
The impact of the concentration and distribution of dissolved and particulate B-vitamins and their congeners on marine microbial ecology
School
College of Letters, Arts and Sciences
Degree
Doctor of Philosophy
Degree Program
Marine Biology and Biological Oceanography
Publication Date
08/30/2018
Defense Date
06/29/2017
Publisher
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Tag
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committee chair
), Berelson, William M. (
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