The chemical form of silicon in marine Synechococcus

Accepted Manuscript The chemical form of silicon in marine Synechococcus

Daniel C. Ohnemus, Jeffrey W. Krause, Mark A. Brzezinski, Jackie L. Collier, Stephen B. Baines, Benjamin S. Twining PII: DOI: Reference:

S0304-4203(18)30037-9 doi:10.1016/j.marchem.2018.08.004 MARCHE 3582

To appear in:

Marine Chemistry

Received date: Revised date: Accepted date:

1 February 2018 6 August 2018 21 August 2018

Please cite this article as: Daniel C. Ohnemus, Jeffrey W. Krause, Mark A. Brzezinski, Jackie L. Collier, Stephen B. Baines, Benjamin S. Twining , The chemical form of silicon in marine Synechococcus. Marche (2018), doi:10.1016/j.marchem.2018.08.004

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ACCEPTED MANUSCRIPT The chemical form of silicon in marine Synechococcus 1 6

Daniel C. Ohnemus*, 2,3 Jeffrey W. Krause, 4 Mark A. Brzezinski, 5 Jackie L. Collier, Stephen B. Baines, 1 Benjamin S. Twining

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Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA Dauphin Island Sea Lab, Dauphin Island, Alabama, USA 3 Department of Marine Sciences, University of South Alabama, Alabama, USA 4 Marine Science Institute and the Department of Ecology Evolution and Marine Biology, University of California, Santa Barbara, California, USA 5 School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, New York, USA 6 Department of Ecology and Evolution, Stony Brook University, Stony Brook, New York, USA

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*Corresponding author: [email protected]

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Abstract The widely distributed marine picocyanobacterium Synechococcus is found throughout the upper oceans. Recent observations that Synechococcus accumulates silicon indicate that it may influence the global cycle of this element. While diatoms, the organisms that dominate the marine Si cycle, have absolute Si requirements due to their precipitation of exoskeleton frustules composed of hydrated opal-A (i.e., biogenic silica), no requirements or biological roles are known for the Si associated with Synechococcus. Even less is known about the biochemical form(s) of Si in picocyanobacteria or whether it differs from diatomaceous silica. Using bulk X-ray absorption near-edge spectroscopy (XANES) across the Si K-edge, we investigated the chemical speciation of Si in dried Synechococcus laboratory isolates from several clades, representing both coastal and oligotrophic isolates grown at a range of Si(OH)4 concentrations. Silicon associated with Synechococcus is bound to oxygen and is spectroscopically distinct from opal-A precipitated by diatoms. The closest XANES spectral analogues found in the literature are dried Mg-Si gels precipitated under basic conditions, suggesting that some Synechococcus Si may be present in vivo as a hydrated siliceous network with Mg and/or Ca cations. Slight spectral variations across isolates and growth conditions suggest strainspecificity in Si chemistry and the potential for siliceous phases to bind organic matter.

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Introduction The discovery of Si accumulation by the abundant and cosmopolitan picocyanobacterium Synechococcus (Baines et al., 2012) has prompted fresh questions about the marine biogeochemistry of this macronutrient element. In diatoms, the dominant marine Si cyclers, Si is precipitated non-stoichiometrically with water as a hydrated amorphous SiO2 glass (biogenic opal-A) during formation of ornate exoskeletal frustules. Complex cellular machinery is dedicated to this process, the biochemistry of which remains an active area of research. Some freshwater cyanobacteria (e.g. Calothrix sp.) found in hot springs are known to spend their entire lives partially or totally encrusted with opal-A that precipitates from ambient fluids supersaturated (i.e. > 2000 µM) with silicic acid (Benning et al., 2004). The oligotrophic surface ocean in which marine Synechococcus cells thrive, however, has sub-saturating ambient silicic acid conditions (typically < 1 µM Si(OH)4 ). Despite lacking the elaborate Si precipitation machinery of diatoms or any known dedicated Si transport system, nearly all marine Synechococcus cells examined via single-cell synchrotron X-ray fluorescence have shown accumulation of 1 – 1000 attomoles (amol, 10–18 mol) Si cell–1 , corresponding to mM intracellular concentrations (Ohnemus et al., 2016). To explain accumulation of this element against such strong concentration gradients, internal precipitation or binding of silicic acid to ligands has been proposed (Brzezinski et al., 2017). A clear physiological role for Si in Synechococcus has not been established, but experiments with a range of laboratory-cultured strains have shown that Si uptake may be associated with acquisition of phosphate, as alleviation of P-stress temporarily reduces Si uptake (Brzezinski et al., 2017). It has thus been suggested that Si accumulation by Synechococcus is inadvertent. Laboratory experiments have also shown that intracellular Si concentrations and uptake rates are strain-dependent (Brzezinski et al., 2017), a result that agrees with the wide range of cell-specific Si quotas observed in mixed natural

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communities (Ohnemus et al., 2016). Experiments have not yet established the chemical form(s) of Si accumulated by Synechococcus, or how it compares to biogenic opal-A that is actively precipitated by diatoms, but culture data suggests there are at least two fractions: Si associated with particulate matter, and Si that is dissolvable in dilute (0.01 M) hydrochloric acid (Brzezinski et al., 2017). While the uptake and accumulation of Si does not appear to directly affect Synechococcus growth rates (Brzezinski et al., 2017), Synechococcus is critically important to larger ecosystem function in the vast oligotrophic oceanic gyres (Sunagawa et al., 2015). It has recently been linked to increased vertical export of carbon from the surface ocean (Guidi et al., 2016; Lomas and Moran, 2011), prompting speculation about whether Si provides slight ballast enhancement. These links have prompted direct investigation of silicon’s chemical form in Synechococcus to better understand their enigmatic utilization of this important macronutrient.

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Outside of the biological realm, Si forms numerous molecular structures with varying degrees of internal polymerization and ordering (Li et al., 1994, 1995). The chemistry of the numerous SiO 2 polymorphs and broader silicate minerals is highly complex due to silicon’s ability to form myriad crystalline (i.e., long-range-ordered) and non-crystalline (i.e., comparatively dis-ordered) molecular structures under various conditions. The basic building block of most environmental silicate oxides, however, is the Si-O tetrahedron, which imparts considerable consistency over short (~2Å) molecular distances from the Si atom. Much of the variability in oxide mineral structures is typically present over medium- (out to 10-20Å) and long-range (> 20Å) distances, where changes in bond angles, tetrahedral interconnection number, and the presence of other bonding partners (e.g. hydration partners and cation substitutions, e.g. Al, Mg, Ca) can occur (Thompson, 2008). Carbon-containing compounds are also known, including silicon-carbides and mixed oxide/carbide phases (e.g. SiO xCyHz; Chaboy et al., 2007). Though these phases are environmentally rare, they cannot be a priori excluded when considering the chemistry of Si structures of unknown biogenic origin.

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To probe the speciation of Si in Synechococcus, we conducted K-edge X-ray absorption spectroscopy in which a core (K-shell) electron from the central Si atom is excited across its absorption edge (energy) by high-flux, tuned X-ray light. Emitted photoelectrons interact with nearby bonding partners, and the resulting absorption spectra, especially near the absorption edge (X-ray absorption near-edge spectroscopy: XANES) can be analyzed both quantitatively and qualitatively. Our primary goals were to establish the major bonding partners of the Si atoms (oxide, carbide, nitrides) in dried cellular material from a range of Synechococcus cultures, to determine whether they differed from diatomaceous opals, and to identify potential phase analogues via qualitative comparison to a selection of mineralogical end-members. These identification efforts represent an important step towards characterization of these unknown biogenic phases, given the large number of potential silicate structures, the fact that the phases in question are unlikely pure specimens, and the sensitivity limits of element-specific analytical techniques when applied to small cells from a complex seawater matrix. Methods

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Synechococcus strains and culturing conditions We examined Si speciation in three Synechococcus strains representing different organismal clades characteristic of a range of marine environments, summarized in Table 1a: CCMP1333 (WH5701; estuarine, Clade 5.3), CCMP1334 (WH7803; low-light, widely-distributed, Clade V) and CCMP2370 (WH8102; ultraoligotrophic, Clade III) (Zwirglmaier et al., 2008). Cultures were procured from the National Center for Marine Algae and Microbiota (NCMA) at the Bigelow Laboratory for Ocean Sciences in East Boothbay, Maine and are referred to using their NCMA (CCMP) number. To examine the effect of ambient silicic acid concentrations, cultures were grown under three conditions (Table 1b): ambient Sargasso seawater (~ 1 µM Si), ambient +60 µM Si(OH)4 , and ambient +120 µM Si(OH)4 , referred to in text and figures using the suffixes -A, -B, and -C, respectively. Strains were grown in polycarbonate culture vessels in surface Sargasso Sea water amended with f/2 nutrients, metals and vitamins, bubbled with 0.2µm filter-sterilized, humidified ambient air to control media pH to prevent the formation of sepiolite, as described in detail by Nelson et al. (1984) and Brzezinski et al. (2017). Temperature was 21˚C and irradiance was 65 µmol photons m–2 s–1 during the daily 12 h light period.

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Log-growth phase cultures were filtered onto 0.2 µm-pore, 25-mm diameter polycarbonate membranes and rinsed with either a seawater-isotonic NaCl solution (artificial seawater; ASW) or distilled water to remove excess media. Filters were transferred to 35 mm petri dishes, covered, and placed in a darkened desiccator for drying at room temperature and storage. Subsequent comparison of ASW-rinsed samples to those rinsed with distilled water indicated no discernable effect of residual salts or rinsing on Si K-edge XANES spectra, so spectra from both rinses were included in the averaged spectra presented here.

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Si K-edge XANES Samples were analyzed at NSLS-I beamline X15B in Brookhaven NY. The beamline utilizes a Si(111) monochromator and is optimized for light-element, “tender” X-ray spectroscopy in the 1-5 keV range. A toroidal mirror focuses the beam to a 1 mm2 spot (<1% of filter area with accumulated cells) with a flux of ~1012 photons s–1 . Spectra were collected via fluorescence and normalized to incident photon flux. The storage ring was operated in “decay mode” which involves re-filling of the ring with electrons every 12 hours; spectra interrupted by storage ring fills were discarded. We averaged three to five scans across the Si K-edge in the energy range 1810-2000 eV (regions beginning at: 1810, 1836, 1850, 1870, 1900, and 1950 eV; steps of 2, 0.1, 0.4, 0.75, 1, and 2 eV, respectively) using 10 s dwell times and 1.8 s settling times per step. Pre-edge removal and post-edge normalization were done using the Athena software package (Ravel and Newville, 2005). Energies were normalized to a quartz mineral standard, with the primary absorption edge maximum (zero of the first derivative) located at 1846.8 eV (Li et al., 1995). Spectra from Li et al. (1995), Thompson et al. (2008), and Ildefonse et al. (1995) were extracted using the WebPlotDigitizer (Rohatgi, 2010) and similarly aligned with the quartz edge of Li et al. (1995) as available. Spectra from Guo et al. (2013) were provided by the authors after Si-wafer and -SiO2 standard edge alignment, a shift of –2

ACCEPTED MANUSCRIPT eV from the originally published spectra. All Synechococcus spectra have had relevant media-blank Si spectra subtracted prior to normalization.

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Mineralogical standards We also collected spectra for a small reference library of Si-containing compounds to provide basic contextual comparison for the unknown siliceous phases: crystalline silicon-carbide (α-SiC), sodium metasilicate (Na2 SiO3 ), α-quartz (α-SiO 2 ), and amorphous hydrated silica (opal-A; SiO 2 ·nH2 O) of the diatom Thalassiosira pseudonana (CCMP 1335). Two diatom treatments were analyzed: whole filtered cells that included both frustules and non-frustuline intracellular Si pools; and acid-cleaned cells (6 M hydrochloric acid at 100˚C for 90 min to remove organic material) that contained frustuline Si only. Whole-cells and HCl-digested diatom treatments displayed visually identical Si K-edge spectra despite known differences in Q 4 /Q3 ratios between frustuline and intracellular Si pools, the latter of which are less condensed (Bertermann et al., 2003). Although these differences are not visually distinguishable in their Si K-edge XANES spectra, for the sake of phase purity only the frustuline Si spectra are averaged and presented here. Additional reference mineral spectra that showed better correspondence to the Synechococcus spectra than those directly collected at the beamline during our experiments were digitized from published references and are included for visual comparison as described above.

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Results Si K-edge XANES spectra of dried cells of Synechococcus clones 1333, 1334, and 2370; diatom frustules (biogenic opal-A glass); quartz; and other reference minerals (discussed further below) are provided in Fig. 1. Silicon fluorescence count rates above the Si Kedge for Synechococcus cultures grown under elevated Si(OH)4 concentrations (‘-B’ and ‘-C’ treatments) were significantly higher than those grown at ambient (1 µM) Si(OH)4 (‘-A’ treatments; only strain 1333 shown). After subtraction of ASW-rinsed growth media filter blanks, the only strain grown at ambient Si(OH)4 that exhibited a meaningful, though still noisy, Si K-edge spectrum was strain 1333 even after averaging of multiple scans. Though many of the lower-Si spectra are noisy, especially when compared to typical published spectra for Si minerals (e.g. Li et al. [1995]), we have shown all strains with recognizable spectra in Fig. 1 due to the paucity of biogenic Si-phase spectra in the literature and to enable their qualitative comparison across a range of Si growth concentrations. The general increase in spectral quality from low-to-high ambient Si is consistent with culture work observations showing that cellular quotas of Si are positively correlated to growth medium Si(OH)4 concentration (Brzezinski et al., 2017), and that Synechococcus Si accumulation varies greatly among strains. The 1333 strain, notably, had the highest cellular Si quotas of the six strains examined in that culture study. In general, material from all cultured Synechococcus strains and Si(OH)4 growth concentrations, but especially the “cleaner” high-Si cultures, presented similar spectral features after the main absorption edge (peak C, Fig. 2), displaying pronounced peaks D, E, and G (Fig. 1), using the nomenclature of Li et al. (1994). All spectra from Synechococcus were notably different from the opal-A spectrum of diatom frustules which had extremely weak peaks D through F or lacked them altogether. Only peaks C

ACCEPTED MANUSCRIPT (the primary absorption edge) and G are notable in spectra from diatom frustules (opalA). Relative expression of intermediate peaks D and E varied across Synechococcus strains and growth conditions: strain 1333 clearly expressed peaks D and E at all Si(OH)4 concentrations, while strains 1334 and 2370 showed these features strongly only when grown at elevated Si.

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Primary edge location, shape, and pre-edge peaks Spectra of strains grown at elevated Si (‘-C’ spectra) exhibited a primary absorption edge (peak C) shifted to lower energy relative to quartz by –0.4 eV (Table 2), compared to – 0.6 eV for 2 of 3 “-B” treatments (Fig. 2). While noisy, spectra from strains grown at low- and intermediate-Si also sometimes displayed a pre-edge peak A, a feature which diminished as strains were grown at higher Si. Strain 2370 expressed an especially clear and strong peak A even at +60 µM Si (“2370-B” treatment), while strain 1333 displayed a very weak peak A at all Si(OH)4 growth concentrations. Pre-edge peaks at the Si-K edge are rare in published literature, but have been observed in laboratory-synthesized Ca-Si hydrates (CSH) where they were enhanced after the addition and putative binding of organic matter (in that case, the drug ibuprofen; CSH+IBU, Fig. 2) by the CSH network (Guo et al., 2013). Pre-edge peak A was not apparent in spectrally similar dried Mg-Si networks from Thompson et al. (2008) that are discussed further below. All Synechococcus spectra and diatoms exhibited primary absorption edges (peak C) that were within 0.6 eV of quartz and without major shoulders (as in SiC) or splitting of the peak (Table 2).

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Comparison with a small mineral reference library and literature spectra Given the wide diversity of potential spectral silicate analogues, our directly-analyzed Si K-edge spectral library was targeted to capture major Si bonding partners: the common silicate oxide mineral, α-quartz; a silicon-carbide mineral end-member, α-SiC; opal-A (a comparatively disordered, hydrated silicate oxide glass) of the cultured marine diatom, Thalassiosira pseudonana; and sodium metasilicate, a sodium silicate salt. Neither diatom frustules nor any of these directly analyzed reference minerals provided good correspondence with the Si K-edge spectra of Synechococcus cultures (Fig. 1).

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After collection of the Synechococcus spectra, several of the most spectrally similar SiO 2 polymorphs (cristobalite, coesite) described by Li et al. (1994) were digitized and compared. Failure of the Synechococcus spectra to match any of the well-characterized SiO2 polymorph end-members prompted an expanded literature search for other Si Kedge spectra, including the major silicate oxide mineral series described by Li et al. (1995). We also digitized Si K-edge spectra of Si-rich allophane (an Al-rich silicate clay mineraloid) and the Al-rich ordered silicate clay kaolinite, as well as nontronite (an Al and Fe-bearing silicate clay), and hisingerite (a ferric-silicate gel) from Ildefonse et al. (1995). A selection of these spectra is shown in Figure 1. Despite the various cation substitutions and degrees of Si polymerization in these references, none of the examined minerals provided convincing spectral matches. We did, however, observe correspondence in peak positions and first-order relative intensities after the main absorption edge (Peak A excepted; Fig 2) with digitized spectra reported for two laboratory-synthesized, solid-phase amorphous Mg-silicate products (Thompson, 2008;

ACCEPTED MANUSCRIPT Thompson et al., 2007). These spectra are shown for more direct comparison to Synechococcus Si and diatom frustules, along with the Mg-rich silicate oxide mineral forsterite analyzed by that group, in Figure 3.

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Discussion Synechococcus maintains surprisingly high dissolved intracellular concentrations of 2 – 24 mM Si via still unknown mechanisms (Brzezinski et al., 2017), compared to typical ambient environmental silicic acid concentrations of ≈ 1 µM (open ocean, oligotrophic) and 10 µM (coastal). These intracellular concentrations should be in significant excess of free silicic acid solubility of ≈ 1 mM, suggesting that most internal Si is either precipitated or bound to ligands, with largely unknown causes or effects. These results— and complementary data showing accumulation of Si by field populations (Krause et al., 2017; Ohnemus et al., 2016) and accumulation of Si- and Mg-rich particles in sinking Synechococcus aggregates (Tang et al., 2014)—have prompted questions about the chemical form or forms of Si present in Synechococcus cells, including whether intracellular Si pools are bound to organic ligands or precipitated as a hydrated gel network and/or more ordered/crystalline solid phase(s). Given how little is known about these particulate phases, it was also important to determine whether they differ substantially from better-studied diatomaceous opals.

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Direct Si bonding environment likely dominated by oxygen The XANES technique probes the average bonding environment of the Si atoms in the sample. Spectra from the Si K-edge can be used to determine major Si-bonding partners, via the location and shape of the primary absorption edge (Peak C, Fig. 2), and to distinguish between comparatively disordered silicate glasses and the numerous ordered silicate minerals, via qualitative examination of spectral shape or comparison to spectra of known mineral end-members. Previous studies have shown that the broad consistency of tetrahedral bond angles and bond lengths imparted by ordered mineral structures generally leads to more pronounced post-edge spectral features (Peaks D-G, Fig. 1). In comparatively disordered glasses these features are diffuse or even absent (Henderson et al., 2014). The energy positions and intensities of XANES peaks can thus be used as a broad fingerprinting tool to determine dominant Si bonding partners, the degree of molecular ordering, and potentially the identity of spectrally charismatic phases. However, the persistence of the well-defined SiO 4 tetrahedron in the structures of many environmental Si oxides also makes the Si K-edge relatively insensitive—compared to other soft elemental edges, e.g. S—to the subtle structural variations present among the numerous potential silicate minerals and glasses (Henderson, 1995). The dominant feature of XANES spectra is the energy position and shape of the primary absorption edge (peak C), which provides insights into the direct bonding partner(s) of the central Si atoms. In cultured Synechococcus, peak C is generally slightly shifted towards lower energy relative to quartz by –0.2 to –0.6 eV, but without significant broadening or splitting of the peak. This suggests the Si-bonding environment is dominated by O—as in quartz, biogenic opal-A and most environmental silicates—since direct replacement of Si-O by Si-C in the tetrahedral structure of SiO x subunits that dominate nearly all natural silicas would likely strongly shift peak C towards lower

ACCEPTED MANUSCRIPT energies if not also significantly broaden or split the peak as observed in α-Si-carbide (Table 2, Fig. 1; shift –1.5 eV). Even partial substitution of Si-O bonds for Si-C or Si-N would likely more strongly shift, broaden, or split the primary absorption peak C, as seen by Chaboy et al. (2007), which is not observed in the Synechococcus samples.

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Diatom frustules also lack the strong energy shifts or splitting of peak C that would indicate direct Si-C or Si-N bonding, suggesting that Si pools of both diatoms and Synechococcus are unlikely to include significant bonding to elements other than O. This is consistent with previous literature examinations of diatom Si processing, in which silicic acid tetrahedral subunits are only loosely coordinated within organic protein structures or ligands and are not direct organically bound (Coradin and Lopez, 2003 and refs. therein).

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The minor energy shifts of the primary absorption edge we observed may indicate that, overall, Si in Synechococcus is less polymerized than in quartz, as shifts of this magnitude and direction have been observed in silicates with lower degrees of internal polymerization of SiO 4 4- subunits (i.e. fewer Q3 and Q4 bonds) (Li et al., 1995). This is further suggested by the fact that 2 of 3 “-B” cultures exhibited lower-energy primary edge positions than the higher-Si “-C” cultures, suggesting an increase in polymerization as cellular Si content increases. Even among known silicate oxide minerals of similar types, however, the scatter in Si K-edge position is known to be broad and thus not unambiguously diagnostic in this regard. More quantitative examination of the degree of Si polymerization using NMR could resolve this ambiguity, ideally utilizing 29 Si-doped cultures to overcome inevitable detection limitations in the complex milieu of biogenic filtered cellular materials.

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Ordering of Synechococcus Si phase(s) and spectral comparisons to Mg-Si gels We observed several intermediate XANES spectral features (peaks D and E) in strain 1333 and others grown at elevated silicic acid concentrations (Fig. 1). These features are thought to be absent in hydrated, biogenic opals (e.g. diatom frustules) due to the absence of medium- to long-range ordering in glasses, which prevents Si-O tetrahedral distortion (Henderson et al., 2014). In more-ordered networks including true minerals, regularity of a molecular matrix leads to arrangements of linked tetrahedra, ranging from small rings and short chains to longer sheets or fully connected 3-D networks (Thompson et al., 2007). These structures impart medium- (2 – 10Å) and longer-range order that strains tetrahedral Si-O orbitals, allowing orbital mixing that is observed as resonances in XANES spectra (Thompson, 2008). The presence of peaks D and E thus suggests some degree of medium-range structural order in the dried siliceous phases of Synechococcus cultures. Our data at the Si K-edge (Fig. 2) are insufficient to unambiguously identify the siliceous phase(s) present or, if multiple phases are present, their relative abundances. However, some initial insights arise from comparison to published Si K-edge spectra of select silicate minerals, SiO 2 polymorphs, and siliceous gel networks. While none of the directly analyzed end-members or published Si K-edge spectra for several SiO 2 polymorphs and Si minerals were clear spectral matches, the positions and relative

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intensities of intermediate peaks D and E of dried Synechococcus Si were most similar to those of un-annealed, amorphous Mg-silicates spontaneously precipitated by Thompson et al. (2007) at mM levels of Si and Mg in weakly basic conditions. While the relative intensities of peaks D and E vary among Synechococcus spectra, the comparative noisiness of our low-Si culture material makes it difficult to determine whether their Product 1 or Product 2 (higher and intermediate Mg content, respectively) is more similar. Qualitatively, however, the separation between peak D and the primary edge (peak C) in their Product 2 is apparent in many of the Synechococcus spectra, suggesting that the lower Mg/Si ratio silicate may be the better analogue. Notably, peak D in dried Synechococcus cultures grown at higher Si is often stronger than in either of the dried, abiotic Mg-silicate gels, instead bearing similarity in prominence to peak D of quartz. This may suggest the presence of a mixture of phases in the dried culture materials: a more crystalline (e.g. quartz-like) phase with a prominent peak D, and a “softer” phase— i.e. a gel containing modifying cation(s), possibly Mg but also potentially Ca or Na, as these cations are also prevalent in the seawater matrix. Notably, the Na-silicate salt we analyzed and other Na-silicate spectra for which Si K-edge XANES are available (e.g. [Henderson, 1995], though not high enough quality to digitize) did not appear to be good spectral matches. However, Ca-rich siliceous phases or mixtures of Mg and Ca phases remain important untested possibilities.

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Given that most sequenced Synechococcus genomes lack known Si-associated genes (i.e. Si transporters, polymerization catalysis proteins, etc.; Collier et al., 2016) and that all cultured Synechococcus isolates examined nevertheless have mM-level intracellular Si(OH)4 concentrations, it is difficult to ignore potential parallels to the spontaneous, amorphous Mg-silicate gel precipitations conducted by Thompson et al. (2007) which produced spectrally similar products. The precipitation conditions of the Thompson et al. Mg-Si gels—millimolar- level Si and Mg concentrations—are not inconsistent with predicted intracellular Si concentrations in cultures and field cells (Brzezinski et al., 2017; Ohnemus et al., 2016). Siliceous gel network precipitation and cation (e.g. Mg, Ca, or Na) incorporation into such networks could potentially occur intracellularly as Synechococcus accumulates Si from its environment, given that intracellular major cation concentrations in marine Synechococcus are similar to seawater and are in the mM range (Mg: 70–138 mM; Ca: 24–90 mM; Na: 90–480 mM; Heldal et al., 2003). Given the potential ambiguity in the Synechococcus phase identification and other sources of uncertainty including the drying process, however, we caution that these parallels cannot yet be interpreted as definitive or mechanistic. To our knowledge, Si phases with structural similarities to cation-containing gel networks have not been previously described in association with soft-tissue marine organisms. However, co-appearance of Mg with Si-rich elemental deposits has been observed in dried, decomposing Synechococcus cells in sinking organic matter from the Sargasso Sea (Tang et al., 2014). Additional characterization of these Si-rich deposits and cultured siliceous phases are required, e.g., through X-ray diffraction as well as X-ray spectroscopy at the Si L-edge or other potential modifying cation edges (e.g. Mg, Ca) to determine their degrees of similarity and to further constrain their compositions. Our own attempts to examine Si-O and Si-Si bond ratios using Raman spectroscopy of dried, bulk

ACCEPTED MANUSCRIPT Synechococcus cultures were frustrated by the presence of residual salts, residual algal pigment complexes, and the polycarbonate membrane substrate (both have considerable C-C and C=C stretching modes) that imparted strong, broadly elevated backgrounds— even in DI-rinsed cultures—that obscured comparatively low-abundance Si Raman peaks.

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Potential speciation differences between in situ and dried phases Our investigations were conducted on rinsed, room-temperature-dried cultures, so we caution that they may structurally differ from the phases actually present in the living organisms. The structures present in or around the organisms in vivo may thus be better described as soft silica gels or gel networks. Many of the Mg-silicates to which our dried phases have the closest spectral similarity were formed in the laboratory by drying siliceous gels that are known to be quite stable while hydrated (Day, 1976; Thompson, 2008).

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We consider here the possibility that the siliceous phases observed are entirely a precipitation artifact of sample preparation, which involves filtration of cells from the media followed by rinsing with seawater-isotonic salt water and drying at room temperature. In their rigorous characterizations of Mg-rich silicate products, Thompson et al. (2007) suggest that hydrated siliceous phases are best described as extended gel networks of oxides and water, using—in the case of their Mg-silicate gels—the generic formulation (MgO)x (SiO 2 )y (H2 O)z. The structure of dried gel products depends greatly on the chemical conditions and speed of the gel’s short-term formation (seconds to minutes) and the conditions of longer-term colloidal aggregation (minutes to days). More rapid reactions between constituents appear to lead to the formation of more crystalline products (Day, 1976). Hydrated gels thus express some poorly-understood degree of imprinting in which the conditions of gel network formation are later reflected in the dried products. However most dried gels do not express significant structural changes in XANES spectra until heating well above 100˚C (Thompson, 2008).

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Relatedly, solid-state 29 Si-NMR of diatom frustule (opal-hydrate glasses) has shown invariance in Q3/Q4 ratios before and after drying at elevated temperatures (100˚C) and longer (12-month) room-temperature storage (Bertermann et al., 2003) than used with our samples. The spectrally-comparable Mg-silicates investigated by Thompson et al. (2007) were themselves initially dried at 75˚C but did not anneal or exhibit significant XANES structural changes until heating above 600˚C. Room-temperature drying of our samples— and also of Si- and Mg-rich materials previously reported in Synechococcus cultures and field aggregates (Tang et al., 2014)—almost certainly alters the speciation somewhat as the gels lose superficial water to form dried, analyzable siliceous products. Nevertheless, multiple lines of evidence suggest that gently-dried products should retain meaningful structural information that reflects associations present in the original, hydrated gel networks without unduly altering the structures of the siliceous phases. Future experiments employing fully hydrated samples are needed to confirm these observations. Strain-to-strain variability and potential network binding of organic matter

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There are differences in spectra among Synechococcus strains grown at lower silicic acid concentrations that are of potential interest. At Si growth concentrations of ≈ 1 µM and 60 µM Si(OH)4 , some Synechococcus strains exhibited a pre-edge feature (peak A) that is rare in mineralogical references (Fig. 2 and Li et al. (1994)). While many of these spectra are noisy, the pre-edge feature was especially prominent in the low-noise “-B” (60 µM Si(OH)4 ) treatment of strain 2370, the most oligotrophic strain examined. In culture studies, the 2370 strain also had the highest mean percent water-soluble Si fraction (determined after four freeze/thaw cycles to lyse cells) of all six strains examined (53 ± 11% vs. ≤ 39 % for strains 1333 and 1334) and was the only strain in which the “-B” treatment had a higher percent soluble Si fraction (69%) than its corresponding “-C” (120 µM Si(OH)4 ) Si treatment (56%) (Brzezinski et al., 2017). If the water-soluble fraction is in some way related to a siliceous phase producing the pre-edge feature (while not differing significantly for other spectral peaks), this could explain why the effect is especially prominent in this particular sample.

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Li et al. (1994) attribute peak A to dipole-forbidden 1s3s transitions that are sometimes more prominent in phases with greater mid-range ordering and thus orbital-mixing than opal-A (e.g. cristobalite, coesite; their Fig. 2), though these minerals did not otherwise match our samples in the rest of the XANES region (our Fig. 1). Interestingly, Guo et al. (2013) have reported the emergence of significant, co-located pre-edge peaks in Si Kedge XANES of several Ca-rich silicate hydrate (CSH) topologies (amorphous, spheres, and nanosheets) after CSH binding to organic matter, in this case the drug ibuprofen. Consistent with Li et al.’s observations and modeling in mineral silicas, Guo et al. attribute enhanced pre-edge peaks in CSH morphologies to increased medium-range ordering of the molecular structure induced by binding of the drug to surface-exposed silanol groups, potentially via esterification with the drug’s carboxylic groups.

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Considering the many potential sites where siliceous phases may form in vivo in Synechococcus (e.g. intracellular interstitial spaces, extracellular polysaccharide matrices), a wide range of organic binding partners may exist, with potential implications for Synechococcus’ interactions with environmental organic matter. The growth of siliceous networks, either inadvertently or directed by these organisms, may have the potential to increase their localized retention of, or adherence to, organic matter via nonspecific binding interactions as observed between Ca-Si-H networks and ibuprofen (Guo et al., 2013). In Synechococcus, pre-edge peak A becomes less apparent but is never entirely absent when strains are grown at higher Si(OH)4 concentrations (“-C” treatments; 120 µM; Fig. 2), suggesting that the siliceous phases exhibiting this spectral feature are less important at higher Si content. We hypothesize that at lower particulate Si concentrations typical of environmental ambient Si(OH)4 , smaller siliceous networks with higher surface-area-to-volume ratios are present in-situ. Associations between the siliceous network and organic molecules could therefore induce a relatively stronger effect on overall Si speciation, leading to a more pronounced peak A. At higher Si(OH)4 concentrations, continued growth of the Si phases should lead to denser siliceous networks and larger particles with lower surface area-to-volume ratios via Ostwald ripening (Williams et al., 1985; Williams and Crerar, 1985). Surficial binding of organic matter to these larger siliceous networks would be less important to overall Si speciation,

ACCEPTED MANUSCRIPT leading to less pronounced Peak A. Of the clones tested, this effect may be most relevant to strains 2370 and 1333 that have higher percent soluble Si fractions. This variability underscores the finding that Synechococcus-associated Si is not homogeneous and that Si chemistry varies across strains and environmental conditions, unlike opal-A that is chemically invariant across the many diatom species precipitating it.

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Extra- or intra-cellular, passive or active formation? Relatedly, and frustratingly, XANES experiments cannot determine if siliceous phases are precipitating extracellularly (i.e. spontaneous precipitation in the Si-enriched culturing media) or intracellularly (i.e. spontaneous or biologically- mediated in an Sienriched internal cell domain). There is a wealth of literature, e.g. for diatoms, describing how organic molecules can control the precipitation and polymerization of biogenic phases. Heldal et al., in their examination of single-cell elemental abundances of Synechococcus cells conclude that internal Mg++ and Ca++ concentrations reflect the cells’ macromolecular content rather than acting as part of the osmoregulatory system. Formation of siliceous networks containing these cations by Synechococcus could be a part of this macromolecular content. Siliceous network formation may occur either in the periplasmic space or within secretions by the organisms—as part of a polysaccharide matrix, for instance—as much remains unknown about the stability, stoichiometry, and physical associations of these biogenic phases.

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Extracellular precipitation could occur in Si-rich conditions that may exist in degrading, sinking particles containing these cyanobacteria. Mg-rich silicates have been reported in association with dried cyanobacterial detritus (Tang et al., 2014), although we did not directly examine such particles to determine their degree of similarity to our cultured cells. Our cells were harvested in mid- to late-exponential phase to avoid any artifacts associated with stationary phase, in which a large proportion of production can be funneled to dissolved organic carbon release.

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Though the molecular stoichiometry and density of the Synechococcus phases are unknown, siliceous gels at mM Si(OH)4 concentrations in these small (< 2 µm diameter) organisms could contribute significantly to cell mass and volume, if not to vertical sinking speed, especially as large numbers of degrading cells aggregate into larger, more rapidly sinking particles (Ohnemus et al., 2016). Given the demonstrated capacity for Carich siliceous phases to bind organic matter (Guo, 2013) and the presence of similar preedge peaks in oligotrophic cultured cellular material, these early results may hint at mechanistic links between siliceous Synechococcus phases and the vertical export of carbon from the euphotic zone (Guidi et al., 2016; Lomas and Moran, 2011). Notably, Synechococcus PCC8806 (which is unrelated to marine Synechococcus) has seen recent utilization in concrete restoration, as incorporation of cyanobacteria into cements enhances the precipitation of calcium carbonate (Zhu et al., 2015). The role that silicification plays in the calcification process is rather controversial (Kremer et al., 2012; Matsko et al., 2011), though the results of Zhu et al. suggest silicification may precede calcification in cyanobacteria. Calcium does not appear to be significantly accumulated in the Mg-rich, EPS-associated marine cyanobacterial particles observed by Tang et al.

ACCEPTED MANUSCRIPT (2014), though our results cannot exclude CSH networks as being part of the in vivo marine Synechococcus siliceous milieu. Industrial (e.g. ceramics, cement) and environmental researchers may find growing overlap in their fields as investigation of these organisms continues.

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Implications for future assessments of biogenic silica in field samples The conclusion reached here that Synechococcus Si is found in different chemical forms than the opal-A glass of diatom frustules prompted suspicion that typical laboratory methods used to determine cellular Si content in cultures and field samples, which utilize leaching with sodium hydroxide (NaOH) (Krause et al., 2013), may under-estimate cell Si content due to their diminished ability to dissolve more-ordered Si phases (Ragueneau and Tréguer, 1994). In natural samples, this is thought to be methodologically desirable: biogenic Si is presumed to be completely digested by the leach while refractory lithogenic minerals, which are only weakly accessed by it, can be accounted for using a minor correction. In a one-off follow-up experiment with strain 1333 (the highest Siaccumulating strain cultured), use of a room-temperature 2.5 M hydrofluoric-acid digestion increased apparent cellular Si quotas by 75% over the standard NaOH leach in a paired comparison. This result appears to support our general observation of moreordered Si phase(s) associated with Synechococcus, either the siliceous gel networks or more crystalline phases indicated by XANES results, and their potentially diminished accessibility to the traditional alkaline leach which was designed for diatom-rich samples. Further experiments with additional isolates are needed to determine if changes to leaching procedures (e.g. different solvents) are warranted when analyzing these organisms or their degradation products.

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The chemical speciation of Si in cultured Synechococcus appears to be markedly different from that found in diatoms, both in low- and high-Si(OH)4 growth conditions. Whether the same siliceous phases are observed in heterogeneous field samples and Si-rich EPS matrices remains to be determined. The presence of Mg- or Ca-silicate phases in association with Synechococcus could, however, be useful in determining the relative influence of cyanobacterial versus diatomaceous Si sources in the field. Depending on how Synechococcus siliceous phases are degraded or weathered as biodetritus sinks, our results suggest that spectral differentiation between diatomaceous Si and cyanobacterial Si may be possible in sinking particle aggregates where Synechococcus-detrital enrichment of particulate biogenic silica (and particulate organic carbon) export is suspected. Such source differentiation using X-ray spectroscopy may also be possible in sediments, again depending on the weathering of the relevant phases and the relative abundance of Si-rich lithogenic interferences. Expanded XANES and NMR analyses of additional sample types, including the Mg-and-Si-rich EPS matrices described at the Bermuda rise (Tang et al., 2014), will be helpful in addressing these questions, as many methodological challenges exist to proper and specific characterization of these phases. Conclusions Si K-edge spectroscopy indicates that Si in Synechococcus is a silicate oxyhydroxide or hydrous oxide(s) that is spectrally distinct from amorphous biogenic opal precipitated by diatoms. While no spectral identification was found among common SiO 2 polymorphs,

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silicate oxide minerals, or aluminosilicate gels, dried Synechococcus spectra were most similar to dried amorphous Mg-silicate gels. These Mg-rich siliceous phases and their hydrated gel network precursors may spontaneously precipitate at the mM levels of Si and Mg and/or Ca present intracellularly or in the immediate environments of degrading cells. The presence of a pre-edge peak in some spectra, especially at lower particulate Si concentrations in some strains, suggests medium-range ordering induced by binding of chemical constituents, potentially non-specific organic matter, to the siliceous network surface. Many of the XANES spectral features observed in Synechococcus are absent in spectra of diatoms likely due to diatoms’ highly-controlled opal precipitation mechanisms and the resultant lack of medium-range ordering, even in dried glasses. Spectra from a range of Synechococcus strains grown under various Si conditions exhibit differences in spectral noisiness and subtle XANES peak intensities, suggesting intrastrain variability in Si abundance and internal structure. Future characterization of these phases should, if possible, investigate the speciation of fully hydrated Synechococcus and related minerals and mineraloids, as well as the XANES speciation at other elemental edges including Mg, Ca, and Al.

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Acknowledgements We thank Paul Northrup at the National Synchrotron Light Source of Brookhaven National Laboratory for his help in data collection at beamline X15B. We also thank several anonymous reviewers for their help in strengthening this manuscript. This research used resources of the National Synchrotron Light Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under contract number DE-AC02-98CH10886. This project was funded by National Science Foundation OCE grants 1131046 to BST; 1335012 to JWK and MAB; and 1131139 to SBB and JLC.

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Figure and Table Captions Figure 1: Si K-edge XANES regions of quartz, diatom frustules, and Synechococcus strains 2370, 1334, and 1333 as measured in this study. Synechococcus “-A” treatments grown at ambient Si(OH)4 ≈ 1 µM; “-B” treatments: ambient + 60 µM Si(OH)4 ; “-C” treatments: ambient + 120 µM Si(OH)4 . Noisier Synechococcus spectra, from cultures grown at lower Si concentrations, are shown in gray. Contrasting silicon carbide (SiC) and sodium silicate spectra are also shown. *The most similar, but non-matching, SiO 2 polymorphs cristobalite and coesite from Li et al. (1994). **The Al-rich mineraloid silicate allophane and Al-rich silicate clay kaolinite from Ildefonse et al (1995). †Ca-Si hydrate (CSH) standard from Guo et al. (2013). Peaks D through G in quartz, and corresponding peaks in diatoms (including weak peak E) and Synechococcus, are labeled after Li et al. (1994, 1995). Figure 2: Si K-(pre-)edge and edge region for SiC, quartz, diatom frustules, Mgsilicate Product 2 † from Thompson (2008), calcium silicate hydrates (CSH) alone and bound to ibuprofen (CSH+IBU) †† from Guo et al. (2013), and Synechococcus strains 2370, 1334, and 1333 (this study). Synechococcus “-A” treatments: ambient Si(OH)4 ≈ 1 µM; “-B” treatments: ambient+60 µM Si(OH)4 ; “-C” treatments: ambient+120 µM Si(OH)4 . For Synechococcus 1333-A, raw data are shown in gray and a three-point

ACCEPTED MANUSCRIPT moving average is overlaid in black. Peaks A (pre-edge) and C (primary edge) are labeled as in Li et al. (1994). Vertical visual guidelines shown are aligned to Peak C of quartz (1846.8 eV) and Peak A of CSH+IBU (1843.8 eV).

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Figure 3: Si K-edge XANES region of quartz, diatom frustules, and dried Synechococcus cells grown at elevated Si(OH)4 (“-C” treatments) from this study. Spectrally similar dried Mg-Si gel Products 1 and 2 as well as forsterite (Mg2 SiO 4 ) and -SiO2 mineral spectra from †Thompson et al. (2008). Visual guidelines set to the energy locations of peaks D, E, and G in quartz are provided and annotated using the peak nomenclature of Li et al. (1994).

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Growth Media [Si(OH)4 ] Ambient Sargasso seawater ≈ 1 µM Si(OH) 4 Ambient + 60 µM Si(OH) 4 Ambient + 120 µM Si(OH) 4

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B: Media Suffix -A -B -C

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Table 1: (a) Synechococcus strain information and (b) media growth conditions examined in this study. Clade, origin and range information from Zwirglmeier et al. (2008). In the text and figures, strains are referred to by their CCMP# plus a suffix reflecting the growth media. A: Strains CCMP# WH# Clade Origin, range 1333 WH5701 5.3 Estuarine 1334 WH7803 V Low-light, widely distributed 2370 WH8102 III Ultraoligotrophic

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Table 2: Peak C (edge) intensity relative to normalized post-edge jump for spectra plotted in Figure 2 and other analyzed Si-minerals and key phases referred to in the text and figures. †: from Thompson et al. (2008); *: from Li et al. (1994). Peak C energy position (zero of the first derivative) is shown relative to quartz, generally increasing from low to high Si. Italics + gray text: noisier spectra that prevent good determination of edge position and/or jump intensity.

SiC-a Quartz Diatom 2370-C 2370-B 1334-C 1334-B 1333-C

Peak C Max Height 1.6 1.8 2.8 2.8 3.1 3.0 3.3 3.0

Shift Relative to Quartz (eV) -1.5 0.0 -0.1 -0.4 -0.6 -0.4 -0.3 -0.4

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3.3 2.0 2.3 2.2 2.2 2.3

-0.6 -0.2 -0.5 -0.3 +0.1 0.0

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ACCEPTED MANUSCRIPT Highlights for: Ohnemus et al., “The chemical form of silicon in marine Synechococcus”

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Si-speciation in three cultured Synechococcus isolates is investigated using Si Kedge XANES Synechococcus Si is present as an oxyhydroxide or hydrous oxide, spectroscopically distinct from diatom opal No exact spectral analogues found; closest matches are cation-rich, hydrated Sigel networks

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