{"paper_id":"2a089eb7-4e65-4537-bc9b-ee74a684bd63","body_text":"1 \nNovel connec)ons between B-vitamins and microbial communi)es 1 \nalong biogeochemical gradients in a large temperate estuary 2 \nMeriel J. Bi,ner1,2,*, Catherine C. Bannon3 §, Elden Rowland3, Gregor Luetzenburg4, Erin M. 3 \nBertrand3, Lasse Riemann1, Ryan W. Paerl5* 4 \n1Marine Biological SecGon, Department of Biology, University of Copenhagen, Helsingør, 5 \nDenmark 6 \n2Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs. Lyngby, 7 \nDenmark 8 \n3Department of Biology, Dalhousie University, Halifax, Nova ScoGa, Canada 9 \n4Geological Survey of Denmark and Greenland (GEUS), Copenhagen, Denmark 10 \n5Department of Marine, Earth, and Atmospheric Sciences, North Carolina State University, 11 \nRaleigh, North Carolina, USA 12 \n§ present address: Max Planck InsGtute for Marine Microbiology, Bremen, Germany 13 \n*Correspondence: mjebi@dtu.dk, rpaerl@ncsu.edu 14 \nOrcid IDs: 15 \nMJB 0000-0002-3798-6315 16 \nCCB 0000-0002-8581-1069 17 \nER 0000-0003-4756-9125  18 \nGL 0000-0001-5443-7572  19 \nEMB 0000-0002-5950-6810  20 \nLR 0000-0001-9207-2543  21 \nRWP 0000-0003-3980-8181 22 \nRunning head: B-vitamin  dynamics  of a temperate estuary 23 \nAuthor contribu8on statement: 24 \nMJB: ConceptualizaGon, Methodology, Formal analysis, InvesGgaGon, Data CuraGon, WriGng – 25 \nOriginal Drae, WriGng – Review & EdiGng, VisualizaGon, Funding AcquisiGon 26 \nCCB: Methodology, InvesGgaGon, Data CuraGon, WriGng – Review & EdiGng 27 \nER: Methodology, InvesGgaGon, Data CuraGon, WriGng – Review & EdiGng 28 \nGL: InvesGgaGon, WriGng – Review & EdiGng, VisualizaGon 29 \nEMB: Methodology, Resources, WriGng – Review & EdiGng, Funding AcquisiGon 30 \nLR: WriGng – Review & EdiGng, Supervision, Funding AcquisiGon 31 \nRWP: ConceptualizaGon, Methodology, InvesGgaGon, Resources, Data CuraGon, WriGng – 32 \nReview & EdiGng, Supervision, Funding AcquisiGon  33 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 2 \nABSTRACT 34 \nAs B -vitamins are organic cofactors required by prokaryoGc and eukaryoGc planktonic 35 \ncells, their availability impact s aquaGc microbial communiGes and associated biogeochemistry. 36 \nContrary to inorganic nutrients, measurements of B -vitamins from brackish systems are scarce 37 \nand relaGonships between B-vitamins and plankton  composiGon  in estuaries are unclear, limiGng 38 \nour understanding of estuary biology in general as well as how B -vitamins are distributed and 39 \ndispersed in marine systems . Here, we quanGfy mulGple B -vitamins and their vitamers  in 40 \nparGculate and dissolved phases , and characterize microbial community composiGon,  across 41 \nfresh to polyhaline zones of the Neuse River Estuary (NRE), N orth Carolina, USA. We uncover 42 \nelevated concentraGons of B-vitamins within the mid -estuary, Chlorophyll a max imum  along with 43 \na unique suite of dissolved B-vitamin  associated with sporadic surges in pico- and microplankton 44 \npopulaGons. The dynamics of both dissolved and parGculate B-vitamin concentraGons in space 45 \nand Gme were striking - from subpicomolar to high picomolar levels observed and strong short-46 \nterm (weeks) variability. We find notable autochtonous B-vitamin producGon in the estuary, but 47 \nwe expect the ability of the system to deliver these micronutrients to the ocean will depend on 48 \nflushing as well as changes in microbial community . We idenGfy vitamin B1,  B12, psB12  49 \n(pseudocobalamin), and B3 as key explanatory variables for change in prokaryoGc and eukaryoGc 50 \nNRE plankton, providing new evidence of B-vitamin  influence upon estuarine plankton 51 \ncommunity composiGon . Our work reveals new complexiGes in B-vitamin producGon and 52 \nconsumpGon within zones of estuaries while underscoring these micronutrients as key drivers of 53 \nmicrobial plankton composiGon .  54 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 3 \nINTRODUCTION 55 \nEstuaries exhibit dynamic hydrological and physical-chemical properGes which alter 56 \nbiology along the river to ocean conGnuum (Bianchi 2007). Microbial (prokaryoGc and eukaryoGc) 57 \ncommuniGes drive biogeochemical cycling and primary producGon in aquaGc environments – 58 \nincluding estuaries – and the diversity and physiology of these microbes can vary notably across 59 \ntemporal and spaGal scales (Peierls et al. 2012; Hall et al. 2013; Wang et al. 2020). Accordingly, 60 \nidenGfying key factors that influence microbial communiGes over space and Gme is of significant 61 \ninterest. Monitoring inorganic (macro)nutrients in aquaGc ecosystems and microbial community 62 \nresponses has been the focus of many research and monitoring efforts (Paerl 2006). For example, 63 \nelevated nitrogen and phosphorus inputs are parGcularly impacmul  to estuarine microbial 64 \ncommuniGes , leading to eutrophicaGon and promoGon of harmful algal blooms (HABs) 65 \n(Wurtsbaugh et al. 2019).  66 \nThe Neuse River Estuary (NRE, North Carolina) is a major tributary of the 2 nd largest 67 \nestuary complex in the USA, the Albemarle Pamlico Estuarine System (APES) , characterized by 68 \nsalinity and nutrient gradients, seasonality, and hydrological variaGon that impact s plankton 69 \nabundance and composiGon along the estuary (Pinckney et al. 1998; Peierls et al. 2012; Hall et 70 \nal. 2013; Gong et al. 2018) . In the NRE, moderate to low river flow leads to biological 71 \nheterogeneity – specifically a Chlorophyll a (Chl a) max mid -estuary and increased planktonic 72 \nbiomass. Similar to other temperate estuary systems, the main phytoplankton groups in NRE are 73 \nchlorophytes, diatoms, dinoflagellates and cryptophytes (Pinckney et al. 1998; Gong et al. 2018, 74 \n2020) with high abundances of cyanobacteria during summer (Gaulke et al. 2010; Hall et al. 2013). 75 \nProkaryoGc plankton community analyses in the NRE have focused on pathogens (e.g. Vibrio spp.) 76 \n(Froelich et al. 2019) and recently cyanobacterial populaGons (Sánchez-Gallego et al. 2025), and 77 \ndominant populaGons are expected to be similar to other temperate estuaries (Wang et al. 2020). 78 \nChanges in inorganic nutrients and/or hydrological properGes (e.g. salinity, discharge) explain 79 \nsome, but not all, of the observed variability in microbial plankton in the NRE (Peierls et al. 2012; 80 \nHall et al. 2013)  and beyond. Micronutrients like tracemetals and B -vitamins are commonly 81 \noverlooked and the impact of B-vitamins on microbial communiGes  within estuary has not been 82 \nrobustly studied. 83 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 4 \nB-vitamins are a group of eight essenGal micronutrients that primarily funcGon as enzyme 84 \ncofactors in cells - but also as anGoxidants or riboswitch ligands (Lukienko et al. 2000; Miranda-85 \nRíos et al. 2001). Vitamin B1 (B1, thiamin), vitamin B2 (B2, riboflavin), vitamin B3 (B3, niacin or 86 \nniacinamide), vitamin B5 (B5, pantothenate), vitamin B6 (B6, pyridoxine) and vitamin B12 (B12, 87 \ncobalamin) are essenGal for most cells – with the excepGon of some organism s lacking B12 88 \nrequiring enzymes altogether (e.g. SAR11 bacterioplankton) (Carini 2013). Most prokaryoGc and 89 \neukaryoGc plankton taxa are auxotrophic (unable to synthesize a vitamin  they require for 90 \nmetabolism  de novo, termed auxotrophs) for one or more B-vitamins (Tang et al. 2010; Sañudo-91 \nWilhelmy et al. 2014; Paerl et al. 2018). As a result, these taxa require an exogenous source of the 92 \nrespecGve vitamin or vitamers (vitamin related com pounds, such as  precursors for de novo  93 \nsynthesis or degradaGon products) to meet cell needs.  94 \nCommon NRE phytoplankton groups are likely to include auxotrophs and taxa with disGnct 95 \nvitamin requirements. Cyanobacteria are B1 prototrophs (organisms capable of de novo syntesis) 96 \nand are hypothesized to be  dominant  summerGme B1 synthesizers of coastal brackish 97 \nenvironments (Sañudo-Wilhelmy et al. 2014; Bi,ner et al. 2024) . Cyanobacteria also produce 98 \npseudocobalamin (psB12), a cobalamin analog that is not biologically available to most  other 99 \nplankton but can be made useful to other plankton groups aeer microbe -mediated chemical 100 \nremodeling (Helliwell et al. 2016; Heal et al. 2017; Bannon et al. 2024b). We hypothesize that the 101 \navailability of  B-vitamins are important drivers of  unresolved microbial plankton variability  as 102 \nseen in other marine systems (Paerl et al. 2018; Joglar et al. 2020; Bannon et al. 2025). There is 103 \nalso evidence of changes in microbial community composiGon during limiGng B -vitamin 104 \ncondiGons, potenGally altering community composiGon of lower trophic levels and leading to 105 \nvitamin deficiency at higher trophic levels (e.g. seabirds, fish) (Balk et al. 2009; Joglar et al. 2020). 106 \nTherefore, quanGficaGon of B-vitamins and vitamers in aquaGc ecosystems is of considerable 107 \ninterest as it potenGally alters producGvity, biomass, and/or the success of specific taxa. Only few 108 \nstudies simultaneously quanGfied mulGple B-vitamins in the dissolved phase (Sañudo-Wilhelmy 109 \net al. 2012; Heal et al. 2014; Suffridge et al. 2017; Bruns et al. 2022, 2023; Bannon et al. 2025), 110 \nand measurements of B -vitamins/vitamers in environmental plankton biomass are even more 111 \nlimited (Suffridge et al. 2017, 2018; Bannon et al. 2025). 112 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 5 \nKnowledge on B-vitamins and vitamers dynamics along the freshwater, brackish to coastal 113 \nocean gradient is lacking, and their connecGon to patchy biology are unknown - contrasGng with 114 \nwell-described and modeled estuary macronutrient dynamics (Twomey et al. 2005). A few studies 115 \nhave quanGfied select B-vitamins /vitamers in a few brackish systems, and shown that their 116 \nconcentraGons vary by two orders of magnitude between systems (Sañudo-Wilhelmy et al. 2006; 117 \nGobler et al. 2007; Koch et al. 2012; Heal et al. 2014; Tovar-Sánchez et al. 2016; Gómez-Consarnau 118 \net al. 2018; Bruns et al. 2022, 2023; Möller et al. 2022; Bi,ner et al. 2024) . While some of this 119 \nobserved variability may be due to analyGcal difficulGes and differences, o verall, data on 120 \nenvironmental concentraGons of vitamin s/vitamers are scarce and the effects of these 121 \nmicronutrients on microbial plankton growth and community dynamics in estuarine systems are 122 \nnot well understood . Thus, it remains unclear whether estuaries could be significant 123 \nallochthonous sources of B-vitamins to the coastal ocean. Understanding these potenGal sources 124 \nis important in a broader biogeochemical context, as the extremely low picomolar concentraGons 125 \nof dissolved B-vitamins in coastal and open ocean may limit microbial growth.  126 \nHere, we leverage substanGal ongoing biogeochemical monitoring within the NRE and 127 \ntackle the following research quesGons: (1) How do B-vitamin concentraGons change short-term 128 \n(weeks) along the salinity gradient of a temperate estuary (fresh to polyhaline)? (2) What are the 129 \nrelaGonships between B-vitamins and size-differenGated planktonic communiGes? To address 130 \nthese quesGons, we measured B -vitamins and vitamers in two parGculate size fracGons 131 \n(picoplankton 0.22 -3 µm; nano - /microplankton 3 -90 µm) and in the dissolved phase using 132 \ntargeted liquid -chromatography mass spectrometry. Simultaneously, we characterized 133 \npicoplankton (0.22-3 µm) and nano-/microplankton (3 -90 µm) communiG es by 16S and 18S rRNA 134 \ngene sequence analyses. We hypothesized that (1) B-vitamin  concentraGons are dynamic on short 135 \nGme scales and exhibit disGnct pa,erns along the estuary  relaGve to macronutrients; and (2) 136 \nplankton community composiGon is significantly impacted by  B-vitamin /vitamer availability , 137 \nespecially B1 and B12 . The results provide a high resoluGon  into the short-term dynamics of 138 \nmulGple B -vitamins and vitamers across phases and along a salinity gradient of a temperate 139 \nestuary. 140 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 6 \nMATERIALS AND METHODS 141 \nSampling 142 \nNear surface water (0.5 m ) was collected on 26 July, 13 and 27 September, and 11 and 25 October 143 \n2021 from staGons NRE0, NRE50, NRE70, NRE100, NRE120, NRE180 in collaboraGon with the 144 \nUniversity of North Carolina at Chapel Hill InsGtute of Marine Sciences (UNC-IMS) Neuse River 145 \nEstuary Modeling and Monitoring Project (MODMON; h,ps://paerllab.web.unc.edu/modmon/ ; 146 \nFig. 1A). NRE160 was sampled once on 11 Oct as weather condiGons did not permit sampling at 147 \nNRE180. Neuse River flow data was obtained from USGS gauge 02091814 near Fort Barnwell 148 \nupstream of the NRE (USGS NaGonal Water InformaGon System Web Interface:  149 \nh,ps://waterdata.usgs.gov/nwis ; SupporGng InformaGon Fig. S1). 150 \n 151 \n 152 \nFig. 1.  Environmental condiGons at estuary sampling sites. Map of NRE staGons sampled and 153 \ndistance along the river from staGon NRE0 is provided in parenthesis, and color gradient shows 154 \nmedian N:P raGo along the estuary from the sampling Gme points (A), based on inorganic nutrient 155 \ndata from the MODMON monitoring program . Gradients in salinity ( B), temperature ( C), 156 \nparGculate organic carbon (POC, D) and dissolved organic carbon (DOC, E) across the staGons and 157 \nGme points sampled . Data may be found in SupporGng InformaGon Data S1.  158 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 7 \nEnvironmental parameters 159 \nTemperature, salinity, and turbidity were measured by YSI 6600 mulG parameter quality 160 \nsonde (Yellow Springs Instruments, Ohio, USA). ParGculate Organic Carbon (POC) was measured 161 \nby Costech ECS 4010 Analyzer as described before (Peierls et al. 2003). Dissolved Organic Carbon 162 \n(DOC) was measured by Shimadzu TOC -5000A Analyzer as previously described (Crosswell et al. 163 \n2012). Extracted Chl a was determined fluorometrically with a Turner Trilogy Fluorometer (Peierls 164 \net al. 2012) . Nitrate/nitrite (NO 3-+NO2-), ammonium (NH 4+), orthophosphate (PO 43-), total 165 \ndissolved nitrogen (TDN) and silica (SiO2) were measured with a Lachat QuickChem 8000 166 \nAutomated Ion Analyzer (Paerl et al. 2010; Peierls and Paerl 2010). DetecGon limits were 0.05 167 \nµM, 0.50 µM, 0.21 µM and 1.17 µM for NO3-+NO2-, NH4+, PO43- and SiO2, respecGvely. Primary 168 \nproducGvity was assessed by light/dark 14C bicarbonate incorporaGon (Paerl 2006; Gaulke et al. 169 \n2010). These measurements are provided in SupporGng InformaGon Data S1. 170 \nBacterial and phytoplankton abundance 171 \nWhole water samples were kept in the dark on ice overnight and then fixed for flow 172 \ncytometry counGng the following day with glutaraldehyde (0.25% final) for 15 min in the dark at 173 \nroom temperature and stored at -80°C (Paerl et al. 2020). Prior to counGng, samples were thawed 174 \nunGl they reached room temperature. Bacterioplankton and phytoplankton abundances were 175 \ndetermined using a Guava EasyCyte HT (Millipore) flow cytometer equipped with red and blue 176 \nexcitaGon lasers. Phytoplankton were counted based on fluorescence (Paerl et al. 2020)  with 177 \naddiGonal gaGng based on forward sca,er to enumerate single cells rather than aggregates 178 \n(SupporGng InformaGon Fig. S2). Bacterioplankton were counted using SYBR Green I staining and 179 \nwithout any heaGng step  (Brussaard et al. 2010) . Abundances are provided in SupporGng 180 \nInformaGon Data S1. 181 \nMetabolite sample collection and analysis 182 \nSampling bo,les, filtraGon units, and collecGon bo,les (amber, HDPE) were cleaned with 183 \n0.1 M HCl, methanol, and MilliQ water. Sampling bo,les were rinsed with sample water, filled 184 \nwith water through a 90 µm Nitex mesh, and stored at near in-situ temperature in the dark. Water 185 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 8 \nwas filtered within 10 h in a dark room through 3.0 µm polycarbonate (Isopore, Millipore) and 186 \n0.20 µm nylon filters (Millipore). The biomass from the nano - and microplankton fracGon that 187 \npassed through the 90 µm mesh and was retained on the 3.0 µm filters was for simplicity defined 188 \nas “microplankton ”, and the biomass fracGon that was sequenGally retained on the 0.2 µm filter 189 \nwas defined as “picoplankton”. Three bo,les of ~250 mL filtrate per staGon were prepared and 190 \nstored at -20°C. 191 \nMetabolite extracGons were conducted in a dark room with a red headlamp as light 192 \nsource. Dissolved metabolites were captured using C18 Solid Phase ExtracGon (SPE; waters, 10 g) 193 \ncolumns and triplicate extracGons were performed for each sample. Filtrates were thawed at 4°C, 194 \npH adjusted to 6.5 and spiked with 13C-thiamin (thiamine-4-methyl-13C-thiazol-5-yl-13C3, Sigma -195 \nAldrich, 75 pM final) . ParGculate metabolites were extracted from 3.0 and 0.20 µm pore -size 196 \nfilters (Heal et al. 2014), and prior to extracGon, vials with sample filters were spiked with 10 pmol 197 \n13C-B1 (4,5,4 -methyl-13C3, 97%; Cambridge Isotope Laboratories), 1 pmol heavy B2 ( 13C4-15N2, 198 \n97%; Cambridge Isotope Laboratories), and 2 pmol cyano-cobalamin  (Fisher BioReagents). Mean 199 \npercent recoveries of 13C-B1 are provided in SupporGng InformaGon Table S1. Eluted metabolites 200 \nwere analyzed using a Dionex UlGmate-3000 LC system coupled to a TSQ QuanGva triple -stage 201 \nquadrupole mass spectrometer (ThermoFisher) operated in selected reacGon monitoring mode. 202 \nMatrix groups included a high salinity grouping ( NRE180, NRE160, NRE120, NRE100 ) and a low 203 \nsalinity grouping (NRE70, NRE50, NRE00), as matrix differences were expected. For each matrix 204 \ngroup limits of detecGon (LOD) and limits of quanGficaGon (LOQ) were determined by calculaGng 205 \n(x3) and (x10) the standard deviaGon of the QC pool run between samples , respecGvely 206 \n(SupporGng InformaGon Data S 2). For further details see (Paerl et al. 2023a)  or addiGonal 207 \nMethods in the SupporGng InformaGon . 208 \nData analysis was adapted from  (Heal et al. 2014; Bannon et al. 2025) . For parGculate 209 \nsamples, the peaks of B1 and B2 were normalized to the stable isotope internal standard to 210 \nreduce variability from the instrument and sample preparaGon. Metabolite measurements were 211 \nexcluded if only one of the two analyGcal injecGons was above the LOD. The mean of the two 212 \nanalyGcal injecGons was calculated before applying percent recoveries. ParGculate B1 was 213 \ncorrected for percent recovery of the stable isotope internal standard as variable recovery was 214 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 9 \nobserved (SupporGng InformaGon Table S1). Dissolved B1 was corrected for percent recovery of 215 \nthe 13C-B1 spike in each sample. Dissolved HMP , HET, FAMP and cHET were corrected for percent 216 \nrecovery previously determined from samples from the estuary (Paerl et al. 2023a), for matrix 217 \ngroups high and low salinity recovery values from staGon NRE180 and staGon NRE0 were applied, 218 \nrespecGvely (SupporGng InformaGon  Table S1). The mean and standard deviaGon were calculated 219 \nfrom biological replicate samples that passed LOD and LOQ, or batch by batch criteria if between 220 \nLOD and LOQ (SupporGng InformaGon Data S3). A few samples were lost during sample 221 \nprocessing or excluded during analysis see SupporGng InformaGon Methods. 222 \nMetabolite data was analyzed and visualized with heatmaps (R package ComplexHeatmap 223 \nv2.20.0), LODs and LOQs were included with their calculated picomolar concentraGon. Metabolite 224 \nconcentraGons (mean of biological replicates) are shown relaGve to the concentraGon range of 225 \neach compound (rows) . Each sampl e (columns) is made up of the different relaGve vitamin 226 \nconcentraGons, referred to here as a vitamin profile, like a B-vitamin “fingerprint“ from that 227 \nsampl e. Columns and rows were clustered based on Euclidean distances corresponding to 228 \ndifferences between samples and relaGve metabolite concentraGons, respecGvely. To analyze the 229 \nphase parGGoning of vitamins, only measurements above LOD/LOQ were included and visualized.  230 \nDNA extraction and sequencing 231 \nSampling bo,les were rinsed with sample water, filled with water through a 90 µm Nitex 232 \nmesh and stored at near in-situ temperature in the dark. Within six hours of sampling , 1 L of water 233 \nwas sequenGally filtered onto a 3.0 µm pore-size membrane (MCE, Millipore) and a 0.22 µm pore-234 \nsize Sterivex filter (PES, Millipore), which were stored at -20°C. DNA was extracted with the 235 \nDNeasy Blood and Tissue kit (Qiagen) with addiGonal lysozyme and proteinase K steps (Bi,ner et 236 \nal. 2024) and quanGfied (Qubit 3.0, Invitrogen).  237 \nParGal 16S and 18S rRNA genes were PCR amplified from both size fracGons (0.22-3.0 µm, 238 \n3-90 µm) with the KAPA HiFi HotStart ReadyMix (Roche) and primer pairs 515F-Y/926R (Parada et 239 \nal. 2016) and a modified version of 565F/964R primers to avoid mismatches with haptophytes 240 \n(Lin et al. 2017), previously used for NRE plankton community analysis (Gong et al. 2020). Primer 241 \nsequences and PCR condiGons are provided in SupporGng InformaGon  Table S2. Triplicate PCR 242 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 10 \nreacGons were pooled, amplicons were indexed and sequenced with MiSeq 2 x 300 bp v3 243 \n(Illumina) at the Rush Genomics and Microbiome Core Facility, Rush University, Chicago, IL, US A 244 \n(Naqib Ankurand Poggi 2018). 245 \nSequence and data analysis 246 \nReads were trimmed with cutadapt (v3.4) (MarGn 2011), quality filtered (16S: F 27 7, R 247 \n240; 18S: F 280, R 250), dereplicated, denoised, read pairs were merged (minOverlap = 100) and 248 \nchimeras removed in DADA2 (v1.22.0) (Callahan et al. 2016). For both 16S and 18S rRNA gene 249 \nfragments, Amplicon Sequence Variants (ASVs) were generated. Taxonomy was assigned to 16S 250 \nASVs with ‘assignTaxonomy’ from DADA2 with the SBDI -curated version (v 7) (Lundin and 251 \nAndersson 2021) of 16S sequences of GTDB (r09-rs220) (Parks et al. 2018; Pascoal et al. 2024). 252 \nTaxonomy to  18S ASVs was assigned with ‘IdTaxa’ from DECIPHER (Murali et al. 2018) with the 253 \nPR2 (v 5.0.0) database (Guillou et al. 2012) . RelaGve abundances of dinoflagellates may be 254 \noveresGmated due to their high 18S rRNA copy number relaGve to diatoms (Gong and Marche‚ 255 \n2019). 256 \nBoth 16S and 18S ASV tables were rarefied (n = 100) to the lowest sequencing depth (16S: 257 \n15,031 reads; 18S: 1,490 reads) with the ‘rrarefy’ funcGon from the vegan package (v2.6.8)  258 \n(Oksanen et al. 2022) , to account for varying sequencing depth in accordance with recent 259 \nrecommendaGons (Schloss 2024). One sample from a 3.0 µm filter from staGon NRE180 from 25 260 \nOct was removed due to insufficient sequencing depth prior to rarefacGon. Further processing 261 \nwas carried out in phyloseq (v1.46.0) (McMurdie and Holmes 2013). Sequences without a domain 262 \nannotaGon and singletons were removed. Empty taxonomic levels were filled by the nearest 263 \nclassified taxonomic level with ‘tax_fix’ from microViz  (v0.12.10) (Barne, et al. 2021) . 264 \nASV abundance data was Hellinger transformed, environmental and metabolite data was 265 \nz-score transformed prior to transformaGon -based Redundancy Analysis (tb -RDA) and R2 -266 \nadjustment with vegan. Explanatory variables  for a constrained ordinaGon were selected by 267 \nforward selecGon by permutaGon (nperm = 999) of residuals under reduced model by 268 \n‘forward.sel’ implemented in adespaGal (Dray et al. 2012). Significance of the constrained tb-RDA, 269 \nthe axes and the explanatory variables were tested with ‘anova.caa’ from the vegan package. 270 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 11 \nPrincipal Coordinate Analyses (PCoA) were performed with vegan on Bray -CurGs distance 271 \nmatrixes of Hellinger transformed abundance data . CorrelaGon analyses were conducted with 272 \nrstaGx (v0.7. 2, A. Kassambara: h,ps://github.com/kassambara/rstaGx ) and visualized with 273 \ncorrplot (v0.92) (Wei and Simko 2021), the lower LOD (SupporGng InformaGon Data S1) was used 274 \nfor measurements below LOD/LOQ. Linear models were fi,ed with the ‘lm’ funcGon implemented 275 \nin R. Data was analyzed and visualized in the R environment (v4. 4.1) with Gdyverse (v2.0.0) 276 \n(Wickham et al. 2019).  277 \nRESULTS 278 \nHydrological and biochemical condi8ons in the estuary 279 \n NRE surface water salinity ranged from 0 to 17 – peaking at staGon NRE180 furthest down 280 \nthe estuary (Fig. 1B). During high river discharge (SupporGng InformaGon Fig. S 1A) a notable 281 \nfreshwater signature occurred further downstream, e.g. periods of salinity ~4 at NRE70 (Fig. 1B). 282 \nDischarge (Neuse River flow) was elevated between the samplings of 11 and 25 October 283 \n(SupporGng InformaGon Fig. S1A). Surface water temperature peaked at 29°C in July and declined 284 \nto 21°C during October (Fig. 1C), showing a seasonal shie from summer to fall.  NOx 285 \nconcentraGons were below detecGon limit  (< 0.05 µM) , except at NRE0 where concentraGons 286 \nreached up to 57 µM on 13 September. Ammoni um  was mostly  below detecGon limit (< 0.50 µM) 287 \nbut was elevated on 11 October (2.3-11.1 µM). Phosphate concentraGons ranged from < 0.21 to 288 \n2.9 µM and showed highe r levels on 13 September. The raGo of nitrogen to phosphate was 289 \ntypically highest (>20) at NRE0 and decreased towards the middle of the estuary (NRE70; Fig. 1 290 \nA), with the execpGon of 25 October. Between 11 and 25 October, inorganic nutrients became 291 \ndepleted at all staGons aeer a period of higher discharge, change was especially pronounced at 292 \nNRE0 (SupporGng InfromaGon Data S1) . Silica concentraGons followed the salinity gradient, with 293 \nhigher concentraGons upstream (SupporGng InformaGon Fig. S 3B). Dissolved organic nitrogen 294 \n(DON) averaged 292 µg N L-1 with periodically higher concentraGons at NRE0 (e.g. 26 Jul 420 µg 295 \nN L-1). Dissolved organic carbon (DOC) ranged from 399 to 754 µM, both minimum and maximum 296 \nwere observed at NRE0 (Fig. 1E). As is typical for the NRE  (Gaulke et al. 2010), max imum  Chl a 297 \noccurred mid -estuary (NRE50, 70) reaching 20-40 µg L -1 (SupporGng InformaGon Fig. S 3D). A 298 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 12 \nnotable excepGon occurred on October 25th, where 90 µg Chl a L-1 occurred in NRE0 surface 299 \nwater coinciding with peaks in turbidity (13.1 NTU), ParGculate Organic Carbon (POC, 3.9 mg C L-300 \n1, Fig. 1D) and primary producGvity (277 mg  C m -3 h-1; SupporGng InformaGon Fig. S3). 301 \nQuantification of dissolved B-vitamins and vitamers 302 \nTwelve B-vitamin s and vitamers were quanGfied from 22 dissolved phase sampl ing events 303 \n(60 samples; full list found in SupporGng InformaGon Data S3 A, Fig. S4). The biologically acGve 304 \nforms of B12 (Ado -B12, Me-B12) and psB12 (Me-psB12) were not detected, as expected due to 305 \nin-situ photodegradaGon into OH-B12 and OH-psB12, respecGvely (Bannon et al. 2025). Dissolved 306 \nOH-B12 ranged from 0.9 pM  up to 3.0 pM (Fig. 2A), and OH -psB12 concentraGons were notably 307 \nlower - approximately half (0.4 -1.4 pM) that of OH -B12 and occasionally below our detecGon 308 \nlimit  (0.3 pM). ConcentraGons of DMB, the alpha ligand of B12, were above the LOD of 2.8 pM 309 \nbut not quanGfiable in most samples. Temporally and spaGally, concentraGons of dissolved B1 310 \nranged from 19 to 74 pM , while concentraGons of B1 vitamers (HMP , AmMP , FAMP , cHET, HET) 311 \nwere lower and ranged from  (below limit of detecGon  - cHET, HMP , AmMP; SupporGng 312 \nInformaGon Data S2A) to 82 pM for FAMP – a B1 degradaGon product.  313 \nDissolved B2 concentraGons (12-96 pM) were comparable to B1  but with a notable 314 \nincrease across the estuary on Oct 25 following high discharge. Similary to B2, B6 was detected 315 \nin all samples and ranged from 7-36 pM (mean 13.7 ± 6 pM). B5 was notably low (< LOD) at higher 316 \nsalinity staGons (NRE100, 120, 180) , with a unique peak of 159 ± 21 pM at NRE180 on 13 317 \nSeptember. With respect to variability , B1 pyrimidine vitamers (HMP , AmMP , FAMP) showed 318 \nmoderate variaGon (2 to 4-fold change) while B5 (159 ± 21 pM peak; 17 ± 14 pM mean; 13 Sep 319 \nNRE180) and cHET (628 ± 153 pM peak; 58 ± 13 pM mean, 27 Sep NRE50)  concentraGons were 320 \nstable, except occasional high peaks (10-fold higher; Fig. 2B). ConcentraGon pa,erns of dissolved 321 \nB1 and OH-B12 clustered together (clustering of rows), but relaGve concentraGons of dissolved 322 \nOH-psB12 concentraGons showed a unique pa,ern compared to the other vitamins with higher 323 \nconcentraGons (∼4-fold) in the lower estuary (NRE100, 120, 180; Fig. 2). ConcentraGon pa,erns 324 \nof dissolved B2, B6 and B3 clustered together and were characterized by a maximum at NRE100 325 \non 25 Oct. This signature of increased dissolved B2 was also detected upstream at NRE70 on 25 326 \nOct. 327 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 13 \n  328 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 14 \nFig. 2. Dissolved B-vitamins and vitamers. Bar plot showing concentraGons of dissolved OH -B12, 329 \nOH-psB12 and B12 lower ligand DMB (A). Upper and lower dashed lines indicate the average LOQ 330 \nand LOD for DMB, respecGvely. The average LOD and LOQ for OH-B12 was 0.9 pM and 3.4 pM, 331 \nrespecGvely. The average LOD and LOQ for OH -psB12 was 0.3 pM and 1.3 pM, respecGvely . 332 \nSamples below LOD are indicated; error bars show ± standard deviaGon of biological replicate 333 \nwater samples.  Heat map of dissolved B -vitamins and vitamer profiles ( B). Rows represent 334 \nconcentraGon pa,erns across samples for each compound and columns represent B -vitamin 335 \nprofiles for each staGon and sampling date (illustrated by the gray boxes). The mean m etabolite 336 \nconcentraGons across water replicates are shown relaGve to the concentraGon range of each 337 \ncompound; clustering is based on Euclidean distances. For measurements below LOD or LOQ, the 338 \nconcentraGon limits are displayed (SupporGng InformaGon  Data S2A). 339 \n 340 \nFreshwater dissolved vitamin samples (NRE0) clustered together  and showed overall 341 \nlower relaGve concentraGons compared to downstream staGons (Fig. 2B). InteresGngly, higher-342 \nsalinity samples from NRE180 clustered with the freshwater staGon cluster and most NRE120 343 \nsamples, whereas brackish mid -estuary staGons (NRE50, NRE70) formed a separate cluster with 344 \nsome NRE100 and NRE120  samples – jointly poinGng to the middle estuary as site of unique 345 \ndissolved vitamin/vitamers composiGon .  346 \nQuan8fica8on of p ar8culate B-vitamins and vitamers 347 \nSixteen B-vitamin  and vitamers were detected from 23 parGculate sampling events in two 348 \nsize fracGons (0.2-3 µm  - 90 samples , 3-90 µm  - 58 samples ; Fig. 3; full list found in SupporGng 349 \nInformaGon Data S3B, C). Mean parGculate B1 concentraGon (0.2-3 µm: 25  ± 18 pM, 3-90 µm: 21  350 \n± 16 pM) and its range (LOD to ∼70 pM) were similar for both size fracGons. HET in the 0.2-3 µm 351 \nsize fracGon was only quanGfiable at five staGons (NRE0, 50, 70, 100, 120) on 25 Oct, following 352 \nhigher discharge, with a maximum  of 3.6 ± 0.6 pM at NRE120. Peak HET concentraGon occurred 353 \nin the 3 -90 µm size fracGon on 25 Oct at NRE120 and NRE0 reaching ∼12.5 pM, otherwise 354 \nconcentraGons were below ~1-2 pM . Traces of cHET were in both size fracGons but not 355 \nquanGfiable due to low signal to noise raGos and low concentraGons . FAMP concentraGon was 356 \nsimilar in both parGculate size fracGons (0.2 -3 µm: 2.8 -18.1 pM, 3 -90 µm: 4.3 -20.2 pM) . In 357 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 15 \ncontrast, HMP was only quanGfiable in select samples from the 3-90 µm size fracGon from the 13 358 \nand 27 Sep with concentraGons of up to 14  ± 7 pM at NRE120 . ContrasGng with dissolved 359 \nconcentraGons, AmMP reached upwards of 65 ± 10 pM (3-90 µm ) at NRE120 and NRE180 on 25 360 \nOct with notable variaGon between sampl es (non-quanGfiable in other samples ; SupporGng 361 \nInformaGon Data S3). 362 \nMean parGculate B2 varied two-fold between size fracGons (0.2-3 µm: 4.3  ± 2.9 pM, 3-90 363 \nµm: 9.0  ± 8.2 pM) with lowest concentraGons (< 2 pM) across the estuary occurring 11 Oct. B3 in 364 \nthe 0.2-3 µm size fracGon was highly dynamic with variaGons of more than 20 -fold ranging from 365 \n14 pM to a of maximum concentraGon of 306 ± 42 pM at NR70 on 27 Sep. In contrast, B3 in the 366 \n3-90 µm size fracGon were less dynamic with a mean of 20  ± 17 pM, excluding a peak 367 \nconcentraGon of 125 pM at NRE0 on 25 Oct. Generally, parGculate B5 in both size fracGons was 368 \nlowest at freshest staGon NRE0 (< 2 pM), except on 25 Oct where a maximum (50 ± 19 pM in the 369 \n3-90 µm size fracGon) was found at NRE0. 370 \nThree forms of B12 (Me -B12: methylcobalamin, Ado -B12: adenosylcobalamin, OH -B12: 371 \nhydroxycobalamin) and two forms of psB12 ( OH-psB12: hydroxy-pseudocobalamin, Me-psB12: 372 \nmethyl-pseudocobalamin) could be resolved in the parGculate size -fracGon, but bioavailable 373 \nforms (Ado -B12, Me-B12, Me-psB12) were oeen below their respecGve LODs, especially in the 3-374 \n90 µm size fracGon. Mean OH-B12 concentraGon was 1.1 ± 0.5 pM and 1.3 ± 0.8 pM in 0.2-3 µm 375 \nand 3-90 µm size fracGon s, respecGvely. The maximum parGculate concentraGon for a ny B12-376 \ncompound was  Ado-B12 at NRE70 on 27 Sep with 7.6  ± 2.8 pM in the 0.2 -3 µm size fracGon, 377 \nhighlighGng a biological acGve form  of B12 can be twice the concentraGon of OH-B12. 378 \nConcentraGons of DMB (mean 2.2 ± 1.4 pM) were patchier than B12 forms  in the 0.2-3 µm size 379 \nfracGon and between the LOD and 1.7 pM in 3 -90 µm size fracGon  (Fig. 3A, SupporGng 380 \nInformaGon Fig. S5) . Notably, on 25 Oct 2021 3.4  ± 0.6 pM Me -psB12 was detected in the 381 \nmicroplankton.  382 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 16 \n 383 \n 384 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 17 \nFig. 3. ParGculate B-vitamins and vitamers. Bar plot showing concentraGons of parGculate 385 \n(0.2-90 µm) total -B12, total-psB12 and B12 lower ligand DMB ( A). Samples where total-psB12 386 \nwas below the limit of detecGon of 0.1 pM is indicated; error bars show ± standard deviaGon of 387 \nbiological replicate water samples. Heat map of size -fracGonated parGculate B-vitamins and 388 \nvitamer profiles (B). Rows represent concentraGon pa,erns across samples for each compound 389 \nand columns represent B -vitamin profiles for each staGon and sampling date. The mean 390 \nm etabolite concentraGons across biomass replicates are shown relaGve to the concentraGon 391 \nrange of each compound; clustering is based on Euclidean distances. For measurements below 392 \nLOD or LOQ, the concentraGon limits are displayed (SupporGng InformaGon  Data S2B, C). 393 \n 394 \nClustering based on Euclidean distances was performed to idenGfy similariGes and 395 \ndifferences across parGculate metabolites, staGons and sampling dates (Fig. 3B). FAMP , OH-B12, 396 \nand B1 exhibited similar variability in concentraGon pa,erns of parGculate vitamin concentraGons 397 \n(relaGve to the concentraGon range of the respecGve compound) across sampling dates, staGons, 398 \nand the two size fracGons (Fig. 3B; clustering of rows in heatmap). These three compounds (FAMP , 399 \nOH-B12, B1) were characterized by occasionally elevated concentraGons, contrasGng to other 400 \ncompounds  (e.g. B2), which had sporadic someGmes 10 -fold higher concentraGons. ParGculate 401 \nvitamin profiles from the two size fracGons did not exhibit clustering by staGon, sampling Gme, 402 \nor size fracGon (Fig. 3B). Only in four instances did vitamin profiles from the two size fracGons at 403 \nthe same staGon and Gme cluster closely together (e.g., NRE0 on 27 Sep and 11 Oct, NRE120 on 404 \n11 Oct, NRE70 on 25 Oct). Overall, parGculate vitamin profiles from a given size fracGon were 405 \nmore similar to profiles from the same size fracGon at other staGons than to the alternate size 406 \nfracGon at the same staGon (e.g. 0.2-3 µm profiles from NRE100 and NRE120 on 11 Oct).  407 \nWhen clustering the parGculate vitamin profiles per size -fracGon separately, the three 408 \nfreshwater (NRE0) vitamin profiles cluster together in the 0.2-3 µm size fracGon as they were 409 \ncharacterized by overall lower concentraGons contrasGng to samples from the middle estuary 410 \n(NRE70, 100) (SupporGng InformaGon Fig. S6). Highest parGculate vitamin concentraGons in the 411 \n0.2-3 µm size fracGon were measured at NRE70 in September. The parGculate vitamin profile of 412 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 18 \nthe 3-90 µm size fracGon at NRE0 from 25 Oct, was unique with peak concentraGons of B5, psB12 413 \nand HET grouping separately (Fig. 3). 414 \nB-vitamins and abio8c estuary condi8ons 415 \nA Kendall’s rank correlaGon analysis was performed for metabolites with each other and 416 \na suite of associated measurements (Fig. 4A, SupporGng InformaGon Fig. S7). To invesGgate if 417 \nrivers could be a source of vitamins to coastal systems, B -vitamin and vitamer concentraGons 418 \nwere analyzed for correlaGons with salinity, and results were vitamin-specific (Fig. 4). Dissolved 419 \nB2 and B6 were negaGvely correlated with salinity. Dissolved OH-psB12 was posiGvely correlated 420 \nwith salinity and higher concentraGons aligned with higher abundances of picocyanobacteria, 421 \nproducers of pseudocobalamin (Bannon et al. 2024b) (Fig. 4B, SupporGng InformaGon Fig. S8). In 422 \nthe 0.2-3 µm parGculate size fracGon B5 and Me-psB12 were posiGvely correlated to salinity. B1 423 \nand B3 within the 3-90 µm parGculate size fracGon were negaGvely correlated with salinity. 424 \nNext, we examined if pa,erns in B-vitamins/vitamers  were connected to pa,erns of  425 \nmeasured dissolved and parGculate nutrients (Fig. 4A). MulGple dissolved and parGculate 426 \nvitamins  (e.g. B1) displayed negaGve correlaGons to dissolved inorganic nitrogen forms, whereas 427 \nmulGple parGculate B -vitamin concentraGons were posiGvely correlated to POC  and DOC 428 \nconcentraGons. ParGculate B-vitamins (B2, B3 and B12) of the 0.2 -3 µm size fracGon were 429 \nposiGvely correlated to DOC concentraGons. B2 was posiGvely correlated to parGculate nitrogen 430 \nand Chl a in the dissolved and both parGculate phases. ParGculate B-vitamins (B1, B2, B3, B5, OH-431 \nB12) from the 3-90 µm size-fracGon showed strong posiGve correlaGons to ParGculate Nitrogen 432 \n(PN) concentraGons. 433 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 19 \n 434 \n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n?\n−0.59\n−0.46\n−0.33\n−0.2\n−0.07\n0.06\n0.18\n0.31\n0.44\n0.57\n0.7SalinityTemperatureTurbiditydissolved O2POCPNDOCDICNH4NO3+NO2PO4SiO2Chlorophyll aPrimary productivityPC\n−SYN\nPE\n−SYN\nPEUKLEUKcell abundance\nB1\ncHET\nHET\nHMP\nFAMP\nB2\nB3\nB5\nB6\nOH−B12\nOH−psB12\nB1\nFAMP\nB2\nB3\nB5\nOH−B12\nAdo−B12\nMe−B12\nOH−psB12\nMe−psB12\nDMB\nB1\nHET\nFAMP\nB2\nB3\nB5\nOH−B12\nOH−psB12\nDMB\nparticulate 3−90 µm particulate 0.2−3 µm dissolved < 0.2 µm\nA\nB\n1−180\n2−70\n2−100\n2−120 2−180\n3−0\n3−50\n3−70\n3−100\n3−120\n3−180\n4−0\n4−50\n4−100\n4−120\n4−180\n5−0\n5−50\n5−70\n5−100\n5−120\n5−180\nR² = 0.51 \np−value = 0.00018\n0.0\n0.5\n1.0\n0 5 10 15\nsalinity\nOH−pseudoB12 (pM)\nPC−SYN+PE−SYN (cells/mL)\n3e+04\n1e+05\n3e+05\n1e+06\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 20 \nFig. 4. CorrelaGon matrix of significant (p < 0.05) Kendall’s rank correlaGons between metabolites 435 \nand environmental, nutrient and biological measurements ( A). Linear relaGonship between 436 \ndissolved OH -psB12 and salinity and corresponding cell abundances of Synechococcus-like 437 \nphytoplankton (B). Numbers next to points indicate sampling and staGon number. ParGculate 438 \norganic carbon (POC), parGculate nitrogen (PN), dissolved organic carbon (DOC), dissolved 439 \ninorganic carbon (DIC) are abbreviated. Abundances of Synechococcus-like phycocyanin (PC)-rich 440 \ncells (PC -SYN), Synechococcus-like phycoerythrin (PE) -rich cells (PE -SYN), picoeukaryoGc 441 \nphytoplankton cells (PEUK), larger eukaryoGc phytoplankton cells (LEUK) and bacterial cell 442 \nabundance (bacterial abundance) were determined by flow cytometry. For abbreviaGon of 443 \nmetabolites names see SupporGng Data S2. Metabolites below LOD or LOQ were replaced by the 444 \nlower LOD value prior to correlaGon analysis. Gray shading indicates the 95% confidence interval 445 \naround the linear regression. 446 \nPar88oning of B -vitamins across dissolved and par8culate phases 447 \nTo invesGgate vitamin connecGvity between the dissolved and parGculate pool, the 448 \nconcentraGons of vitamins from both parGculate size fracGons were summed. RaGos of 449 \nparGculate to dissolved B1, B3 and B5 varied over Gme and fell on both sides of the idenGty line, 450 \na phase parGGoning of 1:1  (Fig. 5A). ParGculate total B12 concentraGons were higher than 451 \ndissolved OH-B12 concentraGons in most samples, whereas concentraGons of B2, FAMP and HET 452 \nwere higher in the dissolved pool than the parGculate pool. When parGculate vitamin/vitamer 453 \nconcentraGons were normalized to POC and dissolved concentraGons to DOC, pa,erns shieed 454 \ntowards higher values in the parGculate size fracGon (SupporGng InformaGon Fig. S9). 455 \nWhen comparing parGculate B-vitamin and vitamer concentraGons between the two size 456 \nfracGons, B1, B2, B5 and FAMP showed temporal dynamics,  but overall measurements fell around 457 \nthe idenGty line, where the concentraGon of a compound would be equal in both size fracGons 458 \n(Fig. 5B). DisGnctly, total-B12, DMB and B3 concentraGons in the 0.2-3 µm size fracGon were 459 \nhigher compared to the 3-90 µm size fracGon, except B3 on 25 Oct at NRE0. HET concentraGons 460 \nwere oeen elevated in the 3-90 µm size fracGon, whereas OH-psB12 were typically higher in the 461 \n0.2-3 µm size fracGon. 462 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 21 \n 463 \n1\n10\n100\n0.1 1.0 10.0 100.0\npM (total particulate)\npM (dissolved)\nsampling dates\n26 Jul\n13 Sep\n27 Sep\n11 Oct\n25 Oct\ncompound\nB1\nHET\nHMP\nFAMP\nB2\nB3\nB5\nOH−B12 vs. total B12\nOH−psB12 vs. total psB12\nA\n0.1\n1.0\n10.0\n100.0\n0.1 1.0 10.0 100.0\npM (0.2−3.0 µm particulate)\npM (3−90 µm particulate)\nsampling dates\n26 Jul\n13 Sep\n27 Sep\n11 Oct\n25 Oct\ncompound\nB1\nHET\nFAMP\nB2\nB3\nB5\ntotal B12\ntotal psB12\nDMB\nB\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 22 \nFig. 5. Sca,er plots of phase parGGoning of B-vitamins and vitamers, dissolved (< 0.2 µm) 464 \nvs. parGculate (0.2-90 µM) ( A), parGculate (3-90 µm) vs. parGculate (0.2 -3.0 µm; B). Axes are 465 \nlog10 scale and show picomolar concentraGons. Shape of points indicates sampling Gme point 466 \nand color corresponds to metabolite compound. Gray dashed line indicates a phase parGGoning 467 \nof 1:1. Points lee of the line indicate an enrichment in the phase of the y-axis while points right 468 \nof the line indicate an enrichment in the phase on the x -axis. Only metabolites with 469 \nmeasurements in both phases were included; measurements below LODs were excluded. 470 \nParGculate total B12 is the sum of Ado -B12, Me-B12 and OH-B12. ParGculate total psB12 is the 471 \nsum of OH -psB12 and Me-psB12.  472 \n 473 \nB1 was the only compound exhibiGng a spaGal pa,ern across the two parGculate pools 474 \n(SupporGng Data 3B, C ). ParGculate B1 concentraGons were spaGally disGnct with higher 475 \nconcentraGons (31 ± 21 pM) in the lower estuary (NRE180, 120, 100) in the 0.2-3 µm size fracGon 476 \ncompared to the 3-90 µm size fracGon (18  ± 17 pM); however, in the upper estuary (NRE0, 50, 477 \n70) B1 was higher in the 3 -90 µm fracGon  (25 ± 13 pM).  The most notable temporal feature 478 \nobserved was a sharp increase in B2, B3, B5, and HET concentraGons in the 3–90 µm size fracGon 479 \ncompared to the 0.2 –3 µm fracGon at NRE0 on 25 Oct Oct relaGve to the other Gme points 480 \nsampled. 481 \nAs an indicator of potenGal connecGons between metabolite pools , we analyzed 482 \ncorrelaGons between quanGfied metabolites with Kendall’s rank correlaGon analysis (SupporGng 483 \nInformaGon Fig. S7). Dissolved B1 was posiGvely correlated with HET, while dissolved B3 posiGvely 484 \ncorrelated with dissolved B5. Most dissolved B-vitamins and vitamers were not correlated to their 485 \nparGculate concentraGon of either size fracGon. In the 0.2-3 µm parGculate size fracGon some 486 \nmetabolites were posiGvely correlated with each other (e.g. B3 and OH-B12) and m etabolites of 487 \nthe two parGculate size fracGons exhibited only few significant (p < 0.05) correlaGons with each 488 \nother. 489 \nPlankton abundances 490 \nFour small phytoplankton morphotypes were detected in NRE surface water based on 491 \nflow cytometry  (SupporGng InformaGon Fig. S 2, S3E-I). Synechococcus-like phycoerythrin (PE)-492 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 23 \nrich (PE -SYN) cells ranged from 7.29 x 10 1 to 1.23 x 10 5 cells mL -1, while Synechococcus-like 493 \nphycocyanin (PC)-rich cells (PC-SYN) were more abundant (2.31 x 104 to 1.62 x 106 cells mL-1). PE-494 \nSYN was more abundant at higher salinity staGons and peaked at NRE180, whereas PC-SYN cells 495 \nwere abundant throughout the estuary, except NRE0. PicoeukaryoGc phytoplankton (PEUK) 496 \nsurged at NRE100 on 25 October, reaching 1.58 x 105 cells mL-1. Abundance of larger eukaryoGc 497 \nphytoplankton cells (LEUK, SupporGng InformaGon Fig. S2; example plot with gates) peaked at 498 \nNRE70 on 27 September with 2.66 x 105 cells mL-1. Bacterioplankton abundance was on average 499 \n1.52 x 10 7 cells mL -1 across staGons and samples , except NRE0, where cell abundances were 500 \ntypically 10 x lower, with 4.24 x 106 cells mL-1. 501 \nEukaryo8c plankton community composi8on 502 \nA total of 1,640 eukaryoGc ASVs were retained for analysis, with 19% unique to the 503 \nmicroplankton size fracGon and 32% unique to the picoplankton size fracGon. The major 504 \neukaryoGc plankton divisions present were Stramenopiles, Alveolata, Cryptophyta and 505 \nChlorophyta (Fig. 6A, SupporGng InformaGon Fig. S8). Stramenopiles were abundant in both size 506 \nfracGons (0.2-3 µm: 22  ± 15% relaGve abundance, 3-90 µm: 34  ± 19% relaGve abundance). The 507 \ndiatom genus Cyclotella was highly abundant, especially in September, accounGng for up to 53% 508 \nand 70% of relaGve abundance in the pico - and microplankton, respecGvely  (SupporGng 509 \nInformaGon Fig. S10), in line with peak cell abundances of the large eukaryote phytoplankton 510 \nmorphotype measured by flow cytometry (up to 2.7 x 10 5 cells/mL; SupporGng InformaGon Fig. 511 \nS3E). The Alveolata subdivision Dinoflagellata showed higher relaGve abundances in the 512 \nmicroplankton size fracGon (0.2-3 µm: 11  ± 11%, 3-90 µm: 26  ± 18%) and contributed up to 63% 513 \nin relaGve abundance, whereas Ciliophora showed similar relaGve abundances in the two size 514 \nfracGons (0.2-3 µm: 10  ± 9%, 3-90 µm: 9  ± 10%) with peak relaGve abundances at NRE0 (mean 25 515 \n± 11%). The Dinoflagellata genera Polykrikos, Levanderina and Gyrodinium were among the top 516 \n10 assigned genera (48% of taxa were not assigned a genus) based on relaGve abundance across 517 \nthe dataset and occurred predominantly in the microplankton. Peak relaGve abundances for 518 \nPolykrikos, Levanderina and Gyrodinium were 42% (NRE180), 32% (NRE70) and 20% (NRE120) on 519 \n26 July, respecGvely (SupporGng InformaGon Fig. S9). 520 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 24 \nThe dominant genus of Cryptophyta was Cryptomonas and occurred across staGons and 521 \nboth size fracGons but showed highest relaGve abundances at staGons NRE50 and NRE70 on 27 522 \nSeptember with 42 and 35% relaGve abundance, respecGvely (SupporGng InformaGon Fig. S11). 523 \nChlorophyta were present in higher relaGve abundances in the picoplankton (33 ± 17%) than in 524 \nthe microplankton (6  ± 3%; Fig. 6A). The genera Bathycoccus (e.g. 57% at NRE180 25 Oct) and 525 \nMicromonas showed higher relaGve abundances in the lower estuary compared to Ostreococcus, 526 \nwhich peaked in the upper estuary (e.g. 46% at NRE50 25 Oct, SupporGng InformaGon Fig. S11). 527 \nSpikes in Fungi occurred in single samples (picoplankton size fracGon), e.g. Opisthokonta (21% 528 \nrelaGve abundance) at NRE100 on 11 October  and Rhizophydiales (13% relaGve abundance) at 529 \nNRE0 on 25 October. 530 \nProkaryo8c plankton community composi8on 531 \nIn total, 4,451 prokaryoGc ASVs were detected, 47% were unique to the microplankton 532 \nsize fracGon, and 15% were unique to the picoplankton size fracGon. Overall, a high number of 533 \n1,484 ASVs (33%) could not be classified beyond the order level.  Within the prokaryoGc 534 \npicoplankton (0.2-3 µm) the bacterial phyla Pseudomonadota (mean 59 ± 13%), AcGnomycetota 535 \n(mean 14 ± 8%), Bacteroidota (average 13 ± 6%) and Cyanobacteriota (average 10 ± 8%) were 536 \ndominant based on relaGve abundance ( SupporGng InformaGon Fig. S 12). Pseudomonadota 537 \n(mean 17 ± 5%) and AcGnomycetota (mean 7 ± 2%) were less abundant in the microplankton (3-538 \n90 µm) size fracGon, instead Cyanobacteria (mean 29  ± 11%) and Bacteroidota (mean 23  ± 8%) 539 \nwere higher in relaGve abundance. 540 \nPelagibacterales were high in relaGve abundance in the picoplankton across the estuary 541 \n(mean 4 4 ± 13%), with lower relaGve abundances detected at NRE0 (mean 21 ± 11%, Fig. 6B, 542 \nSupporGng InformaGon Fig. S13). Four genera of Pelagibacterales dominated the picoplankton, 543 \nPelagibacter, SYDM01 and IMCC9063 were abundant across all brackish staGons, whereas 544 \nFonsibacter was predominantly present at NRE0.  The dominant cyanobacterial genus was 545 \nVulcanococcus (Synechococcus-like) with 15  ± 9% and 8  ± 7% of relaGve abundance in the 546 \nmicroplankton and picoplankton size fracGon, respecGvely. On October 25 at NRE0, the class 547 \nCyanobacteriia contributed 31% of relaGve abundance to the microplankton and the potenGally 548 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 25 \ncyanotoxic cyanobacterial species Planktothrix agardhii accounted for 11.5 % in relaGve 549 \nabundance. 550 \n 551 \nFig. 6. EukaryoGc (A) and prokaryoGc (B) plankton community composiGon based on 18S and 16S 552 \nrRNA genes at staGons NRE0, NRE70 and NRE160/180. Taxonomy shown on division and order 553 \nlevel for 18 and 16S rRNA, respecGvely. The sample NRE180 from the microplankton size fracGon 554 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 26 \nwas removed due to low sequencing depth for the 18S rRNA gene amplicons. Taxonomic profiles 555 \nfor all sampled staGons are provided in SupporGng InformaGon Fig. S10 and S13. 556 \n 557 \nPrincipal Coordinate Analyses (PCoA) of prokaryoGc and eukaryoGc plankton community 558 \nshowed a clustering of NRE0 samples (SupporGng InformaGon Fig. S14). At staGon NRE0 33% and 559 \n18% of the ASVs of eukaryoGc and prokaryoGc plankton were unique, respecGvely. The 560 \nprokaryoGc plankton community at staGon NRE70 in July was more similar to the NRE0 561 \ncommuniGes sampled across summer and fall , in line with lower salinity and higher river 562 \ndischarge (SupporGng InformaGon Fig. S2, S13, S14). Across all samples, community composiGon 563 \nclustered by size fracGon rather than sampling Gme. The prokaryoGc community at mid -estuary 564 \nstaGon NRE50 was usually disGnct compared to up- and downriver staGons. 565 \nConnec8vity between B-vitamins and plankton communi8es 566 \nPotenGal connecGons between B-vitamin/vitamer  pools and the bacterioplankton and 567 \nphytoplankton abundances were examined by correlaGon analysis (Fig. 4, SupporGng InformaGon 568 \nFig. S1 5). Dissolved  OH-psB12 strongly and posiGvely correlated with PC-SYN and PE -SYN 569 \nabundance, addiGonally parGculate OH -psB12 was posiGvely correlated to PC -SYN. Similarly, 570 \ndissolved B1 posiGvely correlated with PC-SYN abundance (Fig. 4, SupporGng InformaGon Fig. 571 \nS8B). Dissolved and parGculate OH-psB12, and parGculate B2 and B5 of the 0.2-3 µm size fracGon 572 \nposiGvely correlated with bacterioplankton abundances. The (pico-)cyanobacterial order PCC-573 \n6307 was posiGvely correlated with dissolved OH-psB12, parGculate B1 and total psB12 of the 574 \npicoplankton size fracGon  (SupporGng InformaGon Fig. S 8, S 15). RelaGve abundances of of 575 \npicocyanobacteria (0.2-3 µm) were addiGonally posiGvely correlated to DMB. The Chlorophyta 576 \ngenus Bathycoccus (0.2-3 µm) was posiGvely correlated to dissolved OH -psB12 and total 577 \nparGculate psB12. In the 3 -90 µm parGculate size fracGon the relaGve abundance of the 578 \nDinoflagellate genus Levanderina was posiGvely correlated to total B12 . In both size fracGons 579 \nPolykrikos was posiGvely correlated to dissolved B1 and B3. 580 \nRedundancy analys is was used  to examine if B-vitamin and vitamer concentraGons 581 \nsignificantly (p < 0.05) helped explain observed variability in plankton community composiGon . 582 \nSelect B-vitamins  and vitamer s together explained 30.6% of variaGon in eukaryoGc plankton 583 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 27 \ncomposiGon and 42% of the observed variance in prokaryoGc plankton (SupporGng InformaGon 584 \nTable S3). Specifically, parGculate B3 and FAMP, and dissolved B1 and OH -psB12 were highly 585 \nsignificant (p ≤ 0.005) explanatory variables for plankton communit y composiGon . AddiGonal 586 \nsignificant explanatory variables for the eukaryoGc plankton community were dissolved B6 and 587 \nparGculate OH-B12. The strongest explanatory variable for the prokaryoGc community was 588 \ndissolved B1 and parGculate B3 for eukaryoGc plankton. Overall, mulGple B-vitamins , B1- and B12-589 \nvitamers showed a staGsGcally significant effect on plankton composiGon . 590 \nSalinity was the main driving environmental variable for eukaryoGc and prokaryoGc 591 \ncommunity composiGon, explaining 6.9 and 8.5%, respecGvely (SupporGng InformaGon Table S3). 592 \nMeasured environmental variables (aside from B-vitamins/vitamers) explained 13.9% and 20.4% 593 \nof the observed variability in eukaryoGc and prokaryoGc plankton community composiGon, 594 \nrespecGvely. In both eukaryoGc and prokaryoGc communiGes, B -vitamin/vitamer concentraGons 595 \ncumulaGvely explained a greater proporGon of variance, compared to hydrological and nutrient 596 \nmeasurements during our summer –fall sampling.  597 \nDISCUSSION 598 \nConcentra8on paZerns of B-vitamins and vitamers 599 \nHere, we uncover elevated concentraGons of B-vitamins mid -estuary (Chl a max region) 600 \nalong with unique dissolved B-vitamin profiles  associated with higher phytoplankton bioma ss. 601 \nKey bacterio- and phytoplankton groups observed and their potenGal role as B-vitamin producers 602 \nor consumers are disscued below and summarized in a conceptual figure in relaGon to the 603 \ndissolved B-vitamins observed across the freshwater to polyhaline gradient of the NRE in summer 604 \nand fall 2021  (Fig. 7). Our study idenGfie s B1, B3 , and B12 compounds as key compounds 605 \ninfluencing prokaryoGc and eukaryoGc plankton communiGes. The simultaneous quanGficaGon 606 \nof the two dissolved B12 forms, OH-B12 and OH-psB12, show that on average OH-psB12 (mean 607 \n0.9 ± 0.3 pM) was at about 47% of the concentraGon of OH -B12 (mean 1.9 ± 0.6 pM ; Fig. 2A ), 608 \nsupporGng the hypothesis that psB12 could be a crucial source for B12-remodellers, especially in 609 \nB12 limited systems. Dissolved OH-psB12 emerged as a key driver of both prokaryoGc and 610 \neukaryoGc community composiGon in the NRE, strengthening its importance as an indirect B12 611 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 28 \nsource even for organisms thought to be unable to remodel the compound. EsGmates suggest up 612 \nto 17% of bacteria can salvage B12 (Shelton et al. 2019), and the geneGc capability to remodel 613 \npsB12 or use other cobalamin intermediates might be of high importance for planktonic 614 \nmicroorganisms in the NRE and beyond. While OH -B12 is expected to be the dominant form of 615 \nB12 in the sun -lit aquaGc environments (Bannon et al. 2024a) , OH -psB12 synthesized by 616 \ncyanobacteria, could be an addiGonal important source of B12 to organisms capable of 617 \nremodeling psB12 (Helliwell et al. 2016) . Since the 1950s/1960s evidence has accumulated 618 \nhighlighGng B12 as an essenGal metabolite for aquaGc microorganisms , however fewer than 40% 619 \nof prokaryotes are predicted to synthesize B12 de novo (Shelton et al. 2019) and only now we are 620 \nbeginning to elucidate microbial sources, cycling, and transformaGons of the disGnct B12 forms 621 \n(Soto et al. 2023; Bannon et al. 2024a; Wienhausen et al. 2024). 622 \n 623 \nFig. 7 Conceptual map of key bacterio- and phytoplankton groups observed and their potenGal 624 \nrole as B-vitamin producers or consumers across the freshwater to polyhaline gradient of the NRE 625 \nin summer and fall 2021. The color gradient of the estuary indicates the relaGve total dissolved 626 \nLow\nHigh\nB-vitamin\nconcentrations\nBurkholderiales\nB1     B12\nPicocyanobacteria (PC-\nSYN, Vulcanococcus sp.)\nB1   psB12\nPicocyanobacteria (PE-\nSYN, Synechococcus sp.)\nB1   psB12\nCyclotella\n(Diatoms)\nB1     B12\nLarger phytoplankton:\nPlanktothrix agardhii \nB1   psB12\nOstreococcus\nB1-vitamers     \nB1\nLevanderina \n(Dinoflagellate)\nB1     B12\nCryptophya\nB1     B12\nPelagibacterales\nB1-vitamers     \nB1\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 29 \nconcentraGons of measured B-vitamins and vitamers. Yellow or blue arrows in the taxa bubbles 627 \nindicate potenGal producGon (parGculate & dissolved) or consumpGon (dissolved) of B -628 \nvitamins/vitamers, respecGvely, based on findings from culture studies and/or the correlaGon 629 \nanalyses presented and discussed here. 630 \n 631 \nTracking of B-vitamins/vitamer dynamics across the parGculate and dissolved phase of an 632 \nestuary shows that some of the compounds produced within an estuary are enriched in the 633 \ndissolved phase compared to the parGculate phase (FAMP , HET, OH -psB12; Fig. 5A). We 634 \nhypothesize that these degradaGon compounds (FAMP , HET) and OH-psB12 are not readily used 635 \nby most bacterio- and phytoplankton groups, due to a lack of sufficiently sensiGve transporter or 636 \nvitamin salvage pathways, and therefore accumulate. These compounds require further salvaging 637 \nand remodeling acGvity to fulfil the cellular vitamin requirement and could provide an advantage 638 \nfor microorganisms with these metabolic pathways (Paerl et al. 2023a). For further discussion on 639 \nB1, B3 and more details on the parGGoning of B-vitamins and vitamers across the pico- and nano-640 \n/microplankton phases and the dissolved phase see the SupporGng InformaGon. 641 \nPuta8ve eukaryo8c microbial sources and transforma8ons of B-vitamins 642 \nOur data highlights a Gght connecGon between dominant phytoplankton and specific 643 \nvitamin profiles in the parGculate pool, while we are sGll working to understand the vitamin 644 \navailability resulGng from algal blooms (and more broadly the exometabolomes of blooms over 645 \nGme and space). The phytoplankton growth in the middle of the estuary includes the biosynthesis 646 \nof a phytoplankton -specific suite of vitamin/vitamers, and these micronutrients in turn can 647 \nsupport growth of auxotrophic phytoplankton, including potenGal HABs, and bacterioplankton. 648 \n The dominant eukaryoGc phytoplankton groups idenGfied (with 18S rRNA gene 649 \nsequencing) and their dynamics are typical for late summer, early fall in the NRE, consisGng of 650 \ndiatoms, dinoflagellates, cryptomonas, and chlorophyta (Gong and Marche‚ 2019; Gong et al. 651 \n2020). The highly abundant Cyclotella (diatom) could have a B12 requirement and is likely 652 \nprototrophic for B1, based on findings of experiments with one isolate (Tang et al. 2010; Bertrand 653 \nand Allen 2012), however B12 auxotrophy has been shown to be strain specific. ContrasGngly, 654 \nCryptophytes can have a requirement for both exogenous B1 and B12 (Tang et al. 2010), making 655 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 30 \nthem likely consumers of B1, B12, and their respecGve vitamers. Thus, surges of Cryptophyta 656 \n(Cryptophytes) in NRE could be governed by the availability of B1 and B12.  Similarly, the 657 \nabundance of Chlorophyta taxa Ostreococcus, Micromonas, Bathycoccus could be regulated by 658 \nthe availability of B1 vitamers, as they are B1 auxotrophs but can salvage B1 from vitamers (Paerl 659 \net al. 2015, 2023b) . Thereby, these taxa simultaneously consume vitamers and potenGally 660 \nfuncGon as a source for ‘regenerated’ vitamin in the upper estuary (NRE50, 70; 25 Oct). 661 \nLevanderina (Dinoflagellate) recurrently surged during summer/fall ( SupporGng 662 \nInformaGon Fig. S9) and, based on elevated microplankton parGculate B1 concentraGons during 663 \nhigher abundances of Levanderina (e.g. 28.1 pM B1 and 21.8% relaGve abundance of Levanderina 664 \nfissa NRE70 25 Oct), these dinoflagellates are potenGal B1 producers and sources of B1 to higher 665 \ntrophic levels. Previously, B1 biosynthesis transcripts ( thiC, thiE) were abundant during a 666 \ndinoflagellate bloom (Levanderina fissa) in the NRE (Gong et al. 2017) , further supporGng that 667 \nsome bloom -forming Dinoflagellates in the NRE are significant B1 producers. 668 \nInsight into B-vitamin cycling along an estuary con8nuum 669 \nOur measurements have revealed several new perspecGves on vitamin cycling within 670 \n(temperate, long residence Gme) estuaries: (1) freshwater input indirectly fuels increased levels 671 \nof vitamins mid -estuary by promoGng algal growth and (2) B-vitamins (dissolved and parGculate) 672 \nare likely uGlized quickly in the lower estuary and transferred to higher tropic levels in the benthos 673 \nor downstream (Fig. 7), similar to macronutrients. During extended periods of high discharge, the 674 \nmid estuary peak of planktonic biomass (Chl a, bacterial abundance, POC) could shie further 675 \ndownstream or not occur and accordingly affect vitamin supply to the larger Pamlico Sound (Paerl 676 \net al. 2010; Hall et al. 2013) . Surprisingly, B-vitamin/vitamer concentraGons were not overtly 677 \nelevated for all compounds in the freshest region of the NRE as hog and poultry operaGons are 678 \nextensive within the Neuse River watershed (Lebo et al. 2012) and both are established sources 679 \nof nutrients and presumably B-vitamins (Lune,a et al. 2022) . 680 \nWhile we find evidence that estuaries can be a source of B -vitamins /vitamers to 681 \ndownstream coastal waters (Fig. 2, 4), the extent of supply is likely dependent on the freshwater 682 \ndischarge and vitamin -specific (Fig. 7), as some B -vitamins or vitamers appear to accumulate in 683 \nthe dissolved phase (e.g. psB12, FAMP, Fig. 5A). These accumulated compounds could funcGon as 684 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 31 \nmore ‘recalcitrant’ vitamin/vitamers for the phytoplankton and bacterioplankton downstream. 685 \nOverall, the oligohaline water of the NRE was lower in B-vitamin s and characterized by disGnct 686 \nplankton and B -vitamin/vitamer profiles compared to the brackish part of the estuary  (see 687 \nSupporGng InformaGon ). Prior work found inverse correlaGons between B-vitamin concentraGon 688 \nand salinity suggesGng rivers and groundwater as sources of B-vitamins to coastal systems (Gobler 689 \net al. 2007). Data from coastal regions influenced by the Amazon (Brazil; dissolved B1 and B6) and 690 \nMoulouya (Morocco; dissolved B1, B2, B6, B12) rivers found that estuaries were not a major 691 \nsource to the coastal ocean (Barada et al. 2013; Tovar-Sánchez et al. 2016). While we find negaGve 692 \ncorrelaGons of dissolved B2 and B6 with salinity, posiGve correlaGons are evident with dissolved 693 \ncompounds (HMP , B5 and OH-psB12), indicaGng that a general pa,ern for dissolved B -vitamins 694 \nwith salinity might not exist  in this system . Importantly, salinity is a driving factor for bacterio- 695 \nand phytoplankton community structure in the NRE, as shown here and in previous studies (Paerl 696 \net al. 2020; Sánchez -Gallego et al. 2025) , thereby salinity indirectly affects B -vitamin 697 \nconcentraGons. Overall, our data indicate that the NRE delivers B -vitamins and vitamers into 698 \nPamlico Sound (massive component of the APES) but the extent is expected to depend on flushing 699 \n(antecedent precipitaGon) and changes in microbial community composiGon.  Moreover, growth 700 \nand proliferaGon of HAB spp. within the Pamlico Sound may be impacted by  B-vitamin/vitamer 701 \ndelivery via the NRE – making the process impacmul  to food web, water quality, and ecosystem 702 \nhealth. 703 \nTemporal dynamics of B-vitamins and vitamers 704 \nB vitamin concentraGons across the estuary were highly dynamic - from sub  picomolar to 705 \nhigh picomolar levels - revealing strong short-term variability driven by synthesis, uptake, and 706 \ndegradaGon processes, as well as sporadic surges in pico- and microplankton populaGons.  Two 707 \nmodes of B -vitamin dynamics were observed: 1) moderate concentraGon dynamics (2 to 4 -fold 708 \nchange) within a compound specific range (e.g. dissolved HMP; parGculate FAMP) including 709 \noccasionally elevated concentraGons and 2) strong changes in concentraGons (10-fold or more) 710 \ncharacterized by occasionally high peaks in concentraGon (e.g. dissolved B5 and cHET; parGculate 711 \nB2; Fig. 3, 4). We argue that the dynamic pa,erns observed with mode 1 reflect oscillaGons 712 \nbetween states of balance/imbalance for the planktonic communiGes due to vitamin/vitamer 713 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 32 \nproducGon and consumpGon, resulGng in slight to moderate fluctuaGons. We hypothesize that 714 \nm ode 2 is driven by the strong rise and fall (or elevated acGvity) of specific planktonic populaGons 715 \nleading to disGnct vitamin profiles (e.g. October 25th NRE0, Planktothrix agardhii, peak parGculate 716 \npsB12). 717 \nDuring a bloom, parGculate B -vitamin and vitamer concentraGons can increase sharply, 718 \npotenGally disproporGonally to changes in the dissolved pool as observed for (microplankton) 719 \nparGculate B-vitamin and vitamer concentraGons at NRE0 on 25 Oct. These sporadic episodes of 720 \nrapid biomass accumulaGon  likely reflect phases, during which intracellular vitamin synthesis (or 721 \nuptake and salvage) dominates over release. As phytoplankton become nutrient -limited or 722 \nphysiologically stressed, however, extracellular release (and leakage) of vitamins can rise, a 723 \npa,ern documented for DOM in both cultures and environmental samples. While the transfer 724 \ninto the dissolved phase might be taxa specific (Sultana et al. 2023) , the observed vitamin 725 \nconcentraGon pa,erns suggest overall minimal transfer of vitamin/vitamer to the communal 726 \ndissolved pool – outside of declines and death. 727 \nB-vitamins/vitamers shape microbial community composi8on  728 \nHere, we find evidence of B -vitamins - especially dissolved B1 and OH -psB12 and 729 \nparGculate B3 and FAMP - as key explanatory variables for both prokaryoGc and eukaryoGc 730 \nplankton estuarine communiGes ( SupporGng InformaGon Table S3). This is congruent with 731 \nwidespread auxotrophy for B1 and B12 and the important role of B3 in cellular metabolism. 732 \nTogether, this builds a greater perspecGve that these compounds are key ‘shapers’ of plankton 733 \ncommuniGes.  The concentraGons of B-vitamins and vitamers collecGvely explained 42% and 31% 734 \nof the observed variance in the prokaryoGc and eukaryoGc planktonic community respecGvely - 735 \nhighlighGng the overall importance of B-vitamins in shaping microbial community composiGon in 736 \nestuarine waters, complementary to previous observaGons from bo,le/mesocosm incubaGons 737 \nthat have demonstrated vitamin -driven changes in plankton communiGes in certain habitats 738 \n(reviewed in: Bertrand & Allen 2012; Joglar et al. 2020). 739 \nIn the NRE, Picocyanobacteria are likely de novo synthesizers of B1 and psB12 and reach 740 \nhigher abundances in brackish waters away from the freshwater endmember of the NRE (e.g. 741 \nNRE0; Paerl et al. 2020) . The detecGon of peak parGculate Me-psB12 (3.4 ± 0.6 pM) in the 742 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 33 \nmicroplankton on 25 October coincides with a high relaGve abundance of the filamentous 743 \ncyanobacteria Planktothrix agardhii in the microplankton size fracGon. Congruently, we found a 744 \nlink between dissolved B1, OH-psB12 concentraGons in the picoplankton , and dissolved OH-745 \npsB12 with the abundance of PC -SYN (Fig. 4 , SupporGng InformaGon Fig. S8, S15) highlighGng 746 \nthem as sources. As dissolved OH -psB12 is not readily available to most  plankton a Gghter 747 \ncoupling between picocyanobacterial abundance and psB12 is observed compared to dissolved 748 \nB1, which is likely rapidly uGlized. Recent field data (Roskilde Fjord Denmark; Northwest AtlanGc) 749 \nalso point to picocyanobacteria as important B1 and psB12 sources (Bannon et al. 2024b; Bi,ner 750 \net al. 2024). These lines of evidence support that picocyanobacterial abundance could funcGon 751 \nas a proxy for psB12 concentraGons, and that psB12 provided by (pico -) cyanobacteria could 752 \nsignificantly promote taxa capable of cobalamin remodeling (Helliwell et al. 2016; Soto et al. 753 \n2023). Salvage of B12 from degraded cobalamin and DMB may represent an important yet 754 \noverlooked pathway in aquaGc systems, as cyanobacteria are already considered key planktonic 755 \ncommunity members  - especially through C and N -fixaGon - their role may extend further as 756 \nproducers of key vitamins such as B1 and psB12.  757 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2026. ; https://doi.org/10.64898/2026.02.26.707256doi: bioRxiv preprint \n\n 34 \nACKNOWLEDGEMENTS 758 \nThis work was supported by Independent Research Fund Denmark (9040-00067B to LR, RWP , and 759 \nAnders F. Andersson). MJB received funding from the European Union’s Horizon 2020 research 760 \nand innovaGon programme under the Marie Skłodowska-Curie grant agreement No 801199. RWP 761 \nacknowledges support from NSF OCE NSF OCE award s Oand 2416286. EMB acknowledges 762 \nsupport from NSERC Discovery Grant RGPIN -2015-05009 and Simons FoundaGon Grants 504183 763 \nand 1001702. We are grateful to Jeremy Braddy and Amy Bartenfelder for assistance with water 764 \ncollecGons. We thank the whole UNC -IMS MODMON team for analyzing and providing 765 \nhydrological, chemical, and biological data. We thank Malcolm Barnard for help with filtraGons. 766 \nWe thank UNC -IMS for providing lab space and local logisGcal support. We thank Anders F. 767 \nAndersson for input on the study. 768 \n 769 \nCONFLICTS OF INTEREST 770 \nNo conflicts of interest.  771 \n 772 \nDATA AVAILABILITY STATEMENT 773 \nThe data that support the findings of this study are openly available in the SupporGng InformaGon 774 \nData S1, S2 and S3 deposited at h,ps://doi.org/10.11583/DTU.31353040 . 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