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 precursors, but are unable to synthesize them (Sañudo-Wilhelmy et al., 2014; Paerl et al., 2015). Despite the known B-vitamin requirements of many climate-relevant eukaryotic phytoplankton, including species of diatoms, dinoflagellates, and coccolithophores (Croft et al., 2006; Tang et al., 2010; Sañudo-Wilhelmy et al., 2014), dissolved concentrations of these compounds are remarkably low, ranging from undetectable to hundreds of picomoles per liter of water (Suárez-Suárez et al., 2011; Sañudo-Wilhelmy et al., 2012; Heal et al., 2014). 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 (Panzeca et al., 2006; Sañudo-Wilhelmy et al., 2006; Gobler et al., 2007; Koch et al., 2011; Bertrand and Allen, 2012; Bertrand et al., 2012, 2013). While the importance of B-vitamins to phytoplankton has been well known for decades (Droop, 1962; Carlucci and Silberna, 1969; Provasoli and Carlucci, 1974; Pinto et al., 2002), 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 marine microbial community composition 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 (Suárez-Suárez et al., 2011; Sañudo-Wilhelmy et al., 2012; Heal et al., 2014). The utilization of our new analytical 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 B₁ and B₁₂ which exhibit significant levels of auxotrophy (obligate requirement) among eukaryotic phytoplankton (Croft et al., 2006; Tang et al., 2010; Sañudo-Wilhelmy et al., 2014). This method quantifies three forms of thiamin: unphosphorylated thiamin (B₁ hereafter), thiamin monophosphate (TMP), thiamin pyrophosphate (TPP); a thiamin precursor, the pyrimidine moiety (4-amino-2-methyl-5-pyrimidinyl methanol, HMP); biotin (B₇); and four forms of cobalamin (B₁₂), adenosylcobalamin (AB₁₂), cyanocobalamin (CB₁₂), hydroxycobalamin (HB₁₂), and methylcobalamin (MB₁₂). In addition to the primary target analytes listed above, this method can be used to quantify riboflavin (B₂), pyridoxal (B₆), 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.

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 B₁, B₇, and CB₁₂ were 3.9 × 10⁻⁵ ± 7.8 × 10⁻⁶, 9.9 × 10⁻⁶ ± 8.1 × 10⁻⁶ and 4.2 × 10⁻⁷ ± 2.9 × 10⁻⁷, respectively (Table 3, Figure 1). The concentrations of these compounds in AND4 were lower than those measured in CC9311, with the differences of the means of B₁, B₇, and CB₁₂ concentrations equaling 3.5 × 10⁻⁵, 7.4 × 10⁻⁶, and 3.6 × 10⁻⁷, respectively (Table 3, Figure 1). Recent research has suggested that cyanobacteria, including CC9311, solely have the ability to synthesize and utilize pseudocobalamins, which potentially makes our observed presence of CB₁₂ within CC9311 anomalous (Tanioka et al., 2009; Helliwell et al., 2016). 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 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; Waldrop et al., 2012; Sañudo-Wilhelmy et al., 2014). 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.

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 3 m at SPOT were 481 ± 204 B₁, 156 ± 103 TMP, 17.6 ± 16.8 TPP, 40.8 ± 14.8 HMP, 129 ± 45.7 B₂, 31.9 ± 9.87 B₇, 5.82 ± 6.53 AB₁₂, 1.54 ± 2.46 CB₁₂, 40.8 ± 14.8 HB₁₂, 3.14 ± 5.02 MB₁₂, and 62.1 ± 30.1 Met. At the Atlantic Station, particulate concentrations (pM) in the mixed layer ranged from 3.53–44.4 B₁, 5.64–46.5 TMP, 1.13–7.85 TPP, 5.91–45.56 HMP, 1.68–8.55 B₇, 0.52–4.53 AB₁₂, undetectable-1.21 CB₁₂, 1.21–9.11 HB₁₂, 0.19–2.46 MB₁₂, and 1.97–16.11 Met (Figure 2, Table 4). SPOT was more eutrophic during our sampling with a mean observed POC concentration of 16.4 μM, whereas POC was observed to range between 1 and 4.5 μM 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 (3 m) in March 2015 were: 3.2 × 10⁻⁵ ± 1.4 × 10⁻⁵ B₁: 1.1 × 10⁻⁵ ± 7.1 × 10⁻⁶ TMP: 1.2 × 10⁻⁶ ± 1.3 × 10⁻⁶ TPP: 3.4 × 10⁻⁵ ± 1.9 × 10⁻⁵ HMP: 8.8 × 10⁻⁶ ± 3.7 × 10⁻⁶ B₂: 2.2 × 10⁻⁶ ± 8.3 × 10⁻⁷ B₇: 4.2 × 10⁻⁷ ± 4.9 × 10⁻⁷ AB₁₂: 1.1 × 10⁻⁷ ± 1.8 × 10⁻⁷ CB₁₂: 2.8 × 10⁻⁶ ± 1.2 × 10⁻⁶ HB₁₂: 2.3 × 10⁻⁷ ± 3.7 × 10⁻⁷ MB₁₂: 4.1 × 10⁻⁶ ± 1.7 × 10⁻⁶ Met: 1POC (Table 3, Figure 1). The observed particulate B-vitamin to POC molar ratios observed at 3 m in the Atlantic were 8.1 × 10⁻⁶ ± 1.6 × 10⁻⁶ B₁: 6.2 × 10⁻⁶ ± 5.9 × 10⁻⁷ TMP: 1.8 × 10⁻⁶ ± 2.3 × 10⁻⁷ TPP: 1.3 × 10⁻⁶ ± 4.2 × 10⁻⁷ HMP: 4.2 × 10⁻⁶ ± 3.3 × 10⁻⁷ B₂: 1.8 × 10⁻⁶ ± 6.7 × 10⁻⁷ B₇: 1.8 × 10⁻⁷ ± 1.4 × 10⁻⁷ AB₁₂: 1.4 × 10⁻⁶ ± 2.0 × 10⁻⁷ HB₁₂: 5.2 × 10⁻⁸ ± 3.8 × 10⁻⁸ MB₁₂: 6.8 × 10⁻⁷ ± 1.6 × 10⁻⁷ Met: 1POC. The observed particulate vitamin concentrations (pM), and the POC normalized values (mole particulate vitamin: mole POC) at 3 m in the Atlantic were lower by as much as two orders of magnitude than those observed at 3 m 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 0.2 to10 μm, 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 community at SPOT) should result in lower microbial growth rates, and therefore likely lower particulate B-vitamin requirements.

Stoichiometric estimations showed that the B-vitamin concentrations measured in both cultures and field-collected samples were, on average, between 1 and 2 orders of magnitude higher than the values reported from the phytoplankton cultures analyzed by Carlucci and Bowes (1972) (Figure 1). 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).

Similarly, our measured particulate B-vitamin concentrations (normalized to POC) are, on average, an order of magnitude higher than published vitamin B₁ and B₁₂ calculated cellular quotas based on uptake rates (Droop, 2007; Tang et al., 2010; Paerl et al., 2015). 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; Tang et al., 2010; Paerl et al., 2015). These published per cell values were normalized to C values using published conversion factors: 20fgC/cell bacteria, 200fgC/cell cyanobacteria, and 138pgC/cell microplankton (Waterbury et al., 1986; Caron et al., 1999). 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 3 m at SPOT and all depths at the Atlantic Station. The mean total thiamin (B₁, 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 2.3 × 10⁻⁵ ± 6.8 × 10⁻⁶ and 2.2 × 10⁻⁶ ± 7.7 × 10⁻⁷, respectively (Tables 3, 4). In comparison, the mean estimated cellular quotas for thiamin and cobalamin obtained from Tang et al. (2010) and Paerl et al. (2015) are 6.3 × 10⁻⁷ ± 8.7 × 10⁻⁷ and 2.4 × 10⁻⁸ ± 5.9 × 10⁻⁸ (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-vitamin uptake rates and direct particulate measurements will be necessary to fully understand these relationships.

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 B₁, 9.8–89.2 TMP, 2.5–77.3 TPP, 2.5–28.3 HMP, 40.1–679.4 B₇, 0.07–3.5 AB₁₂, 0.09–0.8 CB₁₂, 0.2–3.1 HB₁₂, 0.4–12.1 MB₁₂, and 0.1–2.1 Met (Table 4, Figure 2). These concentrations were decoupled from inorganic nutrient concentrations (e.g., PO4−3). 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 the 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 3, Table 5). Values greater than one indicate relative particulate pool enrichment, while values less than one indicate a relative enrichment in the dissolved pool. Vitamins B₁, B₇, and MB₁₂ were found to be enriched exclusively in the dissolved pool with ratios ranging from 0.02 to 0.83, 0.01–0.21, and 0.02–0.89 respectively (Figure 3, Table 5). Only the amino acid methionine was found to be always relatively enriched in the particulate pool with ratio values ranging from 1.23 to 239 (Figure 3, Table 5). Ratio values for all analytes peaked at the DCM and decreased at the nutricline (Figure 3, Table 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 3, Table 5). Increased biological activity at the DCM likely creates this relative particulate vitamin 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 (200 m) 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.

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 C₁₈ resin to preconcentrate B-vitamins from seawater. Analysis for both methods is carried out using a gradient elution using a C₁₈ column, followed by direct detection using a LC/MS. This new analytical protocol is used to quantify vitamins B₁, B₂, B₆, B₇, B₁₂, 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.

The methods described in this paper were developed by CS with support and guidance from LC and SS in laboratory space provided by SS. All experiments were conducted by CS. The paper was written by CS. The paper was reviewed by LC and SS. Funding for this work was provided by grants from NSF (#1435666) to SS.

Financial support for this research has been provided by the National Science Foundation (Biological Oceanography grant #1435666).