Amphiphilic exopolymers from Sagittula stellata induce DOM self-assembly and formation of marine microgels

Marine Chemistry 112 (2008) 11–19

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Marine Chemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a r c h e m

Amphiphilic exopolymers from Sagittula stellata induce DOM self-assembly and formation of marine microgels Yong-Xue Ding a, Wei-Chun Chin b,c,⁎, Anthony Rodriguez d, Chin-Chang Hung e,f, Peter H. Santschi e, Pedro Verdugo d,⁎ a

Friday Harbor Laboratories, University of Washington, 620 University Road, Friday Harbor, Washington, 98250, United States College of Engineering, Florida State University, Tallahassee, Florida, 32310, United States c School of Engineering, University of California, Merced, California, 95344, United States d Department of Bioengineering, Friday Harbor Laboratories, University of Washington, 620 University Road, Friday Harbor, Washington, 98250, United States e Laboratory for Oceanographic and Environmental Research, Department of Marine Sciences and Oceanography, Texas A&M University, Galveston, Texas, 77551, United States f Institute of Marine Environmental Chemistry and Ecology, National Taiwan Ocean University, Keelung, 20224, Taiwan, Republic of China b

a r t i c l e

i n f o

Article history: Received 31 December 2007 Received in revised form 23 May 2008 Accepted 28 May 2008 Available online 9 July 2008 Keywords: EPS DOM self-assembly Microgels Hydrophobic interactions FRET Bacteria

a b s t r a c t The reversible self-assembly of dissolved organic matter (DOM) yields Ca-bonded microscopic gels containing an estimated one thousand step increase of organic matter concentration compared to bulk seawater. Field studies indicate that Ca-bonded microgels concentration in seawater range from 106 to 1012 microgels × L− 1 reaching a corresponding estimated global mass of ∼ 1–100 gigatons (Gt) of organic matter. Although this huge gel pool has far reaching implications for the cycling of carbon and other elements in the World Ocean it still remains largely unexplored. A critical pending question is the role of crosslinkers other than Ca-bonds in DOM assembly. Marine bacteria release amphiphilic exopolymer substances (EPS) that are essential for attachment and that could serve as models to investigate if hydrophobic bonds could also be involved in DOM network formation. Here we show that DOM assembly can be readily induced by nanomolar concentrations (20 µg × L− 1) of hydrophobic exopolymer released by Sagittula stellata (SEP). Consistent with previous studies on hydrophobic properties of SEP our results indicate that SEP-induced DOM network formation exhibit characteristic features of hydrophobic interactions. Although the significance of gel formation by bacterial exopolymer in global carbon balance remains unknown, it offers intriguing hints about foraging strategies of marine bacteria. Bacterial exopolymer could be vital for their survival in oligotrophic environments often containing only micromolar levels of substrate. Release of minute quantities of exopolymer may facilitate the capture and concentration of substrate by forming nutrient-rich DOM networks in the bacteria immediate neighborhood. These studies complement and give further support to the hypothesis that low energy physical interactions could play a pivotal role in DOM assembly further emphasizing the urgent need to investigate the mechanism underlying DOM/gel mass transfer in carbon flux dynamics. © 2008 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding authors. W.C. Chin is to be contacted at Friday Harbor Laboratories, University of Washington, 620 University Road, Friday Harbor, Washington, 98250, United States. Verdugo, Tel.: +1 206 543 5994; fax: +1 206 543 1273. E-mail addresses: [email protected] (W.-C. Chin), verdugo@u. washington.edu (P. Verdugo). 0304-4203/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.marchem.2008.05.003

The global carbon cycle includes DOM as a major reservoir containing moieties that play critical roles in the biogeochemistry of ocean. The total amount of dissolved organic carbon (DOC) in the World Ocean is comparable to the mass of carbon in atmospheric CO2 and only slightly smaller than the amount of carbon in terrestrial biomass and soil humus (Hansell and Carlson, 1998). DOM is mainly composed of

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biopolymers that although present at micromolar concentrations in seawater reach a total mass of ∼ 700 GtC (1 Gt = 1015 g) i.e. ∼700 billion metric tons of carbon. DOM is largely composed of refractory organic moieties (Benner, 2002). Recent findings both in laboratory and field studies indicate that ∼ 10–25% of free DOM biopolymers remain in reversible self-assembly equilibrium forming porous networks of microscopic hydrogels (Chin et al., 1998; Kerner et al., 2003; Ding et al., 2007; Verdugo et al., in press). The transfer of this huge mass of DOM to the gel pool results in the formation of polymer networks containing ∼ 104 step concentration increase of organic matter that has broad potential consequences ranging from metal ion exchange to microbial loop and global climate dynamics (Wells, 1998; Leck and Bigg, 2002; Verdugo et al., 2004). Paramount among those is the relationship of gels with microorganisms. Marine gels represent hot spots of concentrated bacterial substrate and could have critical implications for the understanding of how carbon is cycled in the ocean (Caron et al., 1986; Orellana et al., 2000; Moon et al., 2007; Azam, 1998). However, the dynamics of DOM polymer association and gel formation and its role in substrate availability in seawater still remains largely unexplored (Wells, 1998; Verdugo et al., 2004). The present studies follow up previous reports indicating that the spontaneous self-assembly of DOM polymers is driven mainly by Ca2+ bonds (Chin et al., 1998). However, equally intriguing but less explored is the contribution of hydrophobic interactions in gel formation (Kuhn et al., 1982; De Gennes, 1979, 1990). Among those is the potential role of EPS in DOM assembly. EPS released to the seawater by bacteria and phytoplankton is known to contain amphiphilic moieties (Decho, 1990; Wingender et al., 1999). They play important roles in a broad range of processes that are likely to result from intermolecular association – including particle formation, sedimentation, cycling of dissolved metals (Passow, 2000; Passow et al., 2001; Bhaskar and Bhosle, 2005) – making them good candidates to investigate if hydrophobic interactions can drive DOM polymer assembly and gel formation. In this study we used exopolymers from cultures of Sagittula stellata as a model. The Roseobacter clade that includes the genus Sagittula stellata is one of the major marine groups, typically comprising upwards of 20% of coastal and 15% of mixed-layer ocean bacterioplankton communities. They are well represented across diverse marine habitats; members have been found from coastal to open oceans, from sea ice to sea floor, and to exist as free living, or associated to particles, or in commensally relationships with marine phytoplankton, invertebrates, and vertebrates. There is evidence indicating that bacterial extopolymers in general (Decho, 1990; Wingender et al., 1999) and Sagittula stellata in particular (Alvarado Quiroz et al., 2006; Hung et al., 2005) contain amphiphilic moieties. However, our experimental strategy did not rely on previous observations on chemical hydrophobic species in Sagittula stellata exopolymers (SEP). Instead, we used fluorescence resonance energy transfer (FRET) techniques and hydrophobic fluorescence labeling to directly reveal the presence of hydrophobic domains in SEP. Still, the ultimate objectives of our work were to demonstrate that SEP can induce DOM assembly, that assembly in this instance is independent of ionic bonds, and that it exhibits the characteristic temperature dependence of hydrophobic interaction-driven processes.

2. Materials and methods 2.1. Chemicals Nile Red was used as a hydrophobic probe (Molecular Probes, Inc, Eugene, OR, USA). Ethylenediaminetetraacetic acid (EDTA) from Sigma-Aldrich was used to chelate Ca2+. ASW was prepared in Milli-Q (Millipore) deionised water following a formulation from Marine Biological Laboratory, Woods Hole, Massachusetts (http://www.mbl.edu/BiologicalBulletin/ COMPENDIUM/CompTab3.html), including: 423 mM NaCl, 9 mM KCl, 9.2 mM CaCl2, 22.9 mM MgCl2, 25.5 mM MgSO4, 2.1 mM NaHCO3. The composition of Ca2+-free ASW included: 436.7 mM NaCl, 9 mM KCl, 22.9 mM MgCl2, 25.5 mM MgSO4, 2.1 mM NaHCO3, and 1 mM EGTA. Reagents were purchased from Sigma-Aldrich. 2.2. Seawater collection and pre-treatment Seawater samples were collected from a coastal estuary site in Friday Harbor, Washington (48.54° N, 123.02° W) from November 11–15, 2004. Upon collection, water was gravity filtered through a sterile fiberglass membrane followed by 0.22 µm Millipore filtering (all filters were pre-washed with 0.1 N HCl and rinsed with Milli-Q water to avoid contaminants), treated with 3 mM sodium azide (NaN3) to inhibit microbial activity, and stored in clean sterile glass bottles in the dark at 4 °C. 2.3. Specimen collection Sagittula stellata E37 was obtained from the American Type Culture Collection (ATCC) 700073. (http://www.moore. org/microgenome/ microb_detail_7.asp? id=37). Sagittula E37 belongs to the Phylum Class Order: Proteobacteria Alphaproteobacteria Rhodobacterales; Genus Species Strain: Sagittula stellata. The E37 strain from ATCC was collected from coastal Georgia; it has a genome size of ∼4 Mb, and was isolated using seawater lignin enrichment. 2.4. Isolation, purification and composition of Sagittula stellata exopolymers Polysaccharide-rich exopolymers were extracted and purified from Sagittula cultures by alcohol precipitation, centrifugation, and enzymatic digestions following procedures described elsewhere (Hung et al., 2005; Alvarado Quiroz et al., 2006). Carbohydrates were measured by a modified spectrophotometric method (Hung and Santschi, 2001), which is based upon oxidizing the free reduced sugars with the 2, 4, 6-tripyridyl-s-triazine (TPTZ), followed by spectrophotometric analysis (Myklestad et al., 1997). Proteins were measured according to Smith et al. (1985, 1992), using bovine albumin fraction V protein (BSA) as standards. Exopolymeric substances released by phytoplankton and bacteria are a major component of the marine macromolecular DOC pool (Santschi et al., 1998). They are largely composed of polysaccharide-rich anionic colloidal polymers that are thought to play an important role in the formation of marine macrogels, marine snow, biofilms, and in colloid and trace element scavenging (Verdugo et al., 2004). Our analysis

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of EPS from Sagittula stellata gave a relatively high percentage of protein, i.e., 10% in terms of carbon, 40% carbohydrate, and about 2% uronic acids. Previous reports show that 1–2% protein content is sufficient to impart amphiphilic and emulsifying properties to hydrocolloids such as EPS (Sternström, 1989; Dickinson, 2003). Moreover, depending upon specific fractions, the hydrophobic contact area of EPS from Sagittula stellata was of the order of 7 to 12 2 × molecule− 1 (Schwehr et al. unpubl.), which suggests the presence of hydrophobic moieties and agrees with EPS relatively high protein content. 2.5. Polymer assembly and microgel formation Assembly of DOM polymers was monitored by measuring microgel hydrodynamic diameter using dynamic laser scattering spectroscopy (DLS) following protocols published elsewhere (Chin et al., 1998). Briefly, seawater samples were refiltered through a 0.22-µm Millipore membrane (pre-washed with 0.1 N HCl) and poured directly into five 10 ml scattering cells that were then positioned in the goniometer of a Brookhaven BI-200SM laser spectrometer (Brookhaven Instruments, NY, USA). The autocorrelation function of the scattering intensity fluctuations detected at a 45° angle was processed on line by a Brookhaven BI 9000AT autocorrelator, and particle size distribution was calculated by the CONTIN method (Provencher, 1982). With the exception of experiments to study the effect of temperature on assembly, the assembly of DOM, SEP, or mix solutions of the two were periodically monitored by DLS at 20 °C. Results from each sample were collected in triplicate over a 200 h observation period. Calibration of the DLS spectrometer was conducted using standard suspensions of monodisperse latex microspheres (Polysciences, PA, USA). 2.6. SEP self-assembly Five aliquots containing 20 or 100 µg × L− 1 SEP dissolved in ASW containing 3 mM NaN3 were filtered using 0.22 µm Millipore filters (prewashed with 0.1 N HCl) and poured directly into 10 ml scattering cells. Results were collected in triplicate from each of the 10 samples. 2.7. Effect of SEP on DOM polymer assembly In these experiments 0.22 µm-filtered SEP stock was added to five 10 ml aliquots of 0.22 µm-filtered seawater to reach concentrations of either 20 or 100 µg × L− 1 SEP. The solutions were mixed in the scattering cells and immediately mounted in goniometer of the laser spectrometer. Results were collected in triplicate from each of the 10 samples. 2.8. Effect of divalent cations on SEP-induced DOM assembly Previous observations indicate that spontaneous DOM self-assembly result from the formation of Ca2+ bonds between polyanionic residues present in DOM polymer chains. Withdrawal of Ca2+ ions from seawater by either dialysis or chelation results in failure of DOM self-assembly or in dispersion of previously assembled microgels (Chin et al., 1998). To evaluate the potential role of electrostatic bonding on SEP self-assembly and SEP-induced DOM assembly we monitored their respective assembly kinetics in presence and

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absence of Ca2+ in seawater. Self-assembly of SEP was monitored by DLS in three 10 ml samples at a concentration of 100 µM SEP in Ca-free ASW. SEP-induced DOM assembly was monitored in three 10 ml samples using 0.2 µm-filtered seawater containing 100 µM SEP in presence and absence of 10 mM EDTA. Results were collected in triplicate from each sample. 2.9. Hydrophobic labelling Nile Red dissolved in water or other polar solvents is almost non-fluorescent. However, in non-polar environments, as when bound to hydrophobic residues it undergoes a large fluorescence enhancement (λex = 575 nm; λem = 633 nm). This feature is routinely used to investigate the presence of hydrophobic domains in organic moieties (Hou et al., 2000; Yablon and Schilowitz, 2004). In this experiment, three 10 ml ASW samples were labelled with 13 µM Nile Red. We then measured the fluorescence emission in each sample before and after addition of 100 µg × L− 1 SEP using a Shimadzu spectrofluorophotometer (RF-5000U). Results were collected in triplicate from each sample before and after addition of SEP. 2.10. Fluorescence resonance energy transfer (FRET) FRET is the transfer of energy from the excited state of a donor fluorophore to a close (b100 Å) neighbour acceptor chromophore (Lackovicz, 1999). In this case SEP (λex = 285 nm; λem = 580 nm) is the fluorescent donor and the acceptor is the hydrophobic probe Nile Red which excitation at 575 nm is near the peak of the 580 nm SEP emission. If donor and acceptor lay in close proximity (∼10 nm), when the SEP donor is excited (λex = 285 nm) energy transfer take place; the emission of SEP is quenched and the Nile Red acceptor emission (λem = 633 nm) can be readily detected. The experiment consisted in first conducting readings of fluorescence in three controls to confirm that exciting at 285 nm a 13 µM Nile Red solution in ASW results no emission at its characteristic λem = 633 nm. If FRET takes place, as verified by our data, upon addition of SEP the λem = 633 nm Nile Red (13 µM) drastically increased. Results were collected in triplicate in each sample before and after addition of SEP. 2.11. Temperature dependence of SEP self-assembly Hydrophobic interactions are strongly temperature dependent. Experiments to study temperature dependence of SEP self-assembly were conducted by DLS in five 10 ml samples of SEP. Scattering cells containing 100 µg × L− 1 SEP in ASW were mounted in the temperature-controlled goniometer of the laser spectrometer and particle size was sequentially recorded in triplicate at 4 °C, 20 °C, and 30 °C for 200 h. 2.12. Effect of hydrophobic polystyrene nanoparticles on free DOM polymer self-assembly We used synthetic nanoparticles as models to validate the notion that hydrophobic interactions can indeed induce DOM polymer assembly and microgel formation in 0.22 µm-filtered seawater. Polystyrene nanoparticles (Bangs Laboratories, Inc.

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Fishers, IN, USA) with a mean diameter of 24 nm were thoroughly pre-washed by multiple centrifugations in Milli-Q water to remove detergents. The pellet was subsequently resuspended in ASW at a concentration of ∼1 × 1013 nanoparticles × ml− 1. The suspension was then filtered through 0.22 µm Millipore filter (prewashed with 0.1 N HCl) and titrated directly into 10 ml scattering cells containing 0.2 µm-filtered seawater to reach a final concentration of ∼1 × 1010 nanoparticles× ml− 1. DOM polymer assembly with and without addition of nanoparticles was then monitored over time by DSL. 3. Results Effect of SEP in marine microgels assembly was investigated at SEP concentrations that are typical for exopolymers in seawater (Hung et al., 2003). In these experiments the self-assembly kinetics of SEP and the effect of SEP on DOM assembly was compared to DOM spontaneous self-assembly. As shown earlier, DOM polymeric material in seawater resulting from

Fig. 2. Spontaneous assembly of 100 µg×L− 1 SEP in ASWat different temperatures (open triangles 4 °C, open circles 20 °C, open squares, 30 °C). Each point is the average±SD of 15 outcomes of triplicate measurements in five samples.

Fig. 1. A. Assembly kinetics of DOM polymers in 0.2 µm-filtered seawater (circles) and self-assembly of SEP (20 µg × L− 1) in ASW (triangles) evaluated by DLS. Addition of 20 µg × L− 1 SEP to 0.2 µm-filtered SW increases the rate of DOM assembly yielding gels of ∼ 4–5 µm in 40 rather than 60 h (squares). Each point is the average ± SD of 15 outcomes of triplicate measurements in five samples. B. Assembly kinetics of DOM polymers in 0.2 µm-filtered seawater (circles) and self-assembly of SEP (100 µg × L− 1) in ASW (triangles) monitored by DSL. Addition of 100 µg × L− 1 to 0.2 µm-filtered seawater results in quick DOM assembly that reaches equilibrium in ∼10 h yielding microgels of ∼4–5 µm hydrodynamic diameter (squares). Each point is the average ± SD of 15 outcomes of triplicate measurements in five samples.

Fig. 3. A. Self-assembly of 100 µg×L−1 SEP in either ASW (open circles), Ca2+-free ASW (open inverted triangles), or Ca2+/Mg2+-free ASW (open triangles) do not exhibit significant statistical differences. Each point is the average±SD of 15 outcomes of triplicate measurements in five samples. B. SEP-induced (100 µg×L−1) assembly kinetics of DOM polymers in 0.2 µm-filtered seawater in absence (open circles) or presence of 10 mM Ca2+ chelator EDTA (open squares), does not exhibit significant statistical differences. Each point is the average±SD of 15 outcomes of triplicate measurements by DSL in five samples.

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Fig. 4. Nile Red (13 μM) in ASW excited at 575 nm emits at 633 nm, addition of 100 µg × L− 1 SEP increases Nile Red emission 4–5 folds. Fluorescence intensity is expressed as % change in photon counts taking as 100% the count emission of Nile Red before addition of SEP. The bars correspond to average ± SD of 9 outcomes of triplicate measurements in three samples.

0.22 µm filtering can self-assemble reaching equilibrium at 3–5 µm diameter microgels in 50–60 h (Fig. 1A). Assembly follows characteristic second order kinetics that as shown earlier results from a two steps process consisting of formation of nanogels that then anneal together forming microgels (Verdugo, 2007). As expected for surfactant-like amphiphilic moieties (De Gennes, 1990; Kuhn et al., 1998), we found that SEP at a concentration of 20 µg × L− 1 (300 nM-C) can undergo self-assembly in artificial seawater (ASW). It follows a slow kinetics that yields 2–4 µm diameter microgels in ∼200 h. Addition of SEP (20 µg × L− 1) to 0.22 µm-filtered seawater accelerates DOM self-assembly shortening the time to equilibrium from ∼60 to ∼40 h (Fig. 1A). Increasing the concentration of SEP to 100 µg × L− 1 results in acceleration of both SEP self-assembly that reach equilibrium in ∼100 h (Fig. 1B), and in a striking rate increase of DOM assembly that resembles a first order kinetics producing microgels that reach a 4–5 µm equilibrium in less than 10 h (Fig. 1B). SEP self-assembly exhibits the characteristic non-linear temperature dependence of hydrophobic processes, with complete inhibition at 4 °C and a significant increase of the rate of microgel formation between 20 and 30 °C (Fig. 2). We previously demonstrated that DOM self-assembly results from the formation of electrostatic Ca2+ bonds between polyanionic sites present in DOM polymers. Removal of Ca2+ by dialysis or by chelation by ethylenediaminetetraacetic acid (EDTA) abolishes DOM polymer assembly and microgel formation and can readily disperse assembled microgels (Chin et al., 1998). Conversely, assembly resulting from hydrophobic interactions are immune to

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Fig. 6. Addition of hydrophobic polystyrene nanoparticles (1 × 1010 × ml− 1) to 0.2 µm-filtered seawater significantly increases the rate of assembly of DOM (squares) compared to spontaneous DOM self-assemble controls (circles). The bars correspond to average ± SD of 9 outcomes of triplicate measurements in three samples. withdrawal of Ca2+. As shown in Fig. 3A and B, both SEP-induced assembly of DOM or SEP self-assembly remain virtually unchanged in Ca2+-free seawater. Nile Red is a nonpolar benzophenoxazone fluorescence probe consisting of solvatochromic molecule that contains a rigid aromatic group and an exocyclic diethylamine group, with fluorescence spectra (λex = 575; λem = 633 nm). However, Nile Red fluorescence depends on the polarity of solvent and changes drastically with variation of local surrounding hydrophobicity (Yablon and Schilowitz, 2004). In ASW Nile Red (13 µM) exhibits very weak emission that increases ∼4–5 folds upon addition of 100 µg × L− 1 SEP to ASW (Fig. 4). These results are consistent with the idea that hydrophobic domains are indeed present in SEP. Controls for FRET in Fig. 5 show that 285 nm excitation produces negligible 633 nm emission from ASW containing 13 µM Nile Red in ASW. However, increasing SEP (λex = 285; λem = 580 nm) concentration to 25 or 100 µg × L− 1 while maintaining 285 nm excitation produces a characteristic increase of 633 nm Nile Red emission that is proportional to the concentration of SEP. These results confirm the existence of FRET between SEP emission (λem = 580 nm) and Nile Red excitation spectra (λex = 575 nm). This outcome reveals that Nile Red must bind to hydrophobic sites that lay in close proximity to the emission site or sites of SEP. Addition of polystyrene hydrophobic microspheres induced pronounced acceleration of DOM-polymers assembly that mimics the effects of SEP (Fig. 6).

4. Discussion

Fig. 5. SEP excited at λex = 285 nm emits at λem = 580 nm, making it a convenient fluorophore donor for FRET when using Nile Red (λex = 575 nm, λem = 633 nm) as acceptor fluorophore. Addition of 25–100 µg × L− 1 SEP to a 13 mM solution of Nile Red in ASW results in a proportional increase of Nile Red emission at λem = 633 nm (open circles). Fluorescence intensity is expressed as % change of photon counts taking as 100% the background count before addition of SEP.

The discovery that DOM can spontaneously and reversibly assemble forming microscopic gels (Chin et al., 1998) opens an intriguing new field of inquiry in marine sciences. Recent observations confirm unambiguously that native microgels identical to those that spontaneously assemble in the laboratory are also present in untreated samples of seawater collected from 10–4000 m transects (Ding et al., 2007; Verdugo et al., in press). Marine gels may represent a major pool of bioactive elements at the Earth's surface. Results to date (Chin et al., 1998; Ding et al., 2007) suggest that at equilibrium ∼10% of seawater DOM remains in reversible assembly forming microscopic gels. If the studied seawaters are typical of the global DOM pool (7×1017 gC), then up to 7×1016 g (∼70 billion metric tons) of organic carbon may occur as microgels in the ocean. This mass, which does not include any contribution by coexisting nanogels, exceeds the estimated total biomass of ∼4×1015 g of carbon present in marine organisms (Begon et al., 1996) by more than one order of magnitude. The rates at which bioactive elements pass through

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the marine microgel (and nanogel) pools are unknown. However, the similar day-to-week time scales for microgel formation (Chin et al.,1998) and 234Th pumping from colloidal to particulate size (Honeyman and Santschi, 1989; Moran and Buesseler, 1992) suggest that the corresponding fluxes could be huge. These observations emphasize the critical significance of DOM assembly and the urgent need to understand the molecular mechanisms responsible for gel formation in the ocean. Polymer networks can form physical or chemical gels. Physical gels are made up of organic molecules connected physically by low energy ionic forces, hydrophobic linkages, or entanglement. Chemical gels are made of polymer chains crosslinked by high energy covalent bonds; once polymer chains are crosslinked the assembly process of these networks is irreversible and their size depends primarily upon the concentration of both polymer chains and crosslinker. In networks like marine microgels held together by tangles and low energy interaction polymer concentration affects the kinetics of assembly. However, their average equilibrium size results from the dynamics of the assembly-dispersion equilibrium, i. e. from interconnections and tangles that are continuously making and braking. The assembly-dispersion dynamics of tangled networks like DOM or ESP-assembled microgels depends primarily on the second power of the ensemble average of chain length of the polymers that make them. This is because in order for polymer to leave the gel they needs to randomly rept (axially diffuse) its way out of the network. Since diffusion times depend on the second power of the length of the diffusional random walk, the stability of tangled networks that determines the equilibrium size of the gels is critically determined by chain length (de Gennes and Leger, 1982; Doi and Edwards, 1984). Low energy interactions are an important additional factor that contributes to the stability of tangled gels. In networks made out of polyelectrolyte chains, charge density and counter ion concentration contribute to stabilize the matrix by electrostatic interactions. In amphiphilic networks the ratio of hydrophobic/hydrophilic domains present in the polymer chains and the concentration of short chain amphilite are responsible for the hydrophobic interactions that are important factors in initiating the formation and keeping tangled networks together. Nonetheless, it is important to keep in mind that low energy interactions do not provide stable crosslinks since they are continuously making and breaking; chain length remains the critical parameter that determine the stability and final equilibrium size in these gels. Thus, the reason why microgels don't grow bigger than 4– 5 µm is most likely because the average polymer chain length of DOM is rather limited and as their size grows they become more susceptible to dispersive forces driven by diffusional or convectional shear. This is very well illustrated by the fact that at constant DOM concentration UV-cracking of DOM polymers results in longer assembly times and smaller gels; at the limit, long time short wave UV exposure results in complete inhibition of microgel formation (Orellana and Verdugo, 2003). In here we investigate the role of the second most important group of low energy interactions that could produce intermolecular DOM crosslinking, in this case by formation of hydrophobic bonds. Among the potentially most active species inducing DOM assembly is the stock of bacterial EPS.

These amphiphilic moieties are widely distributed and have been thought to play an important role in particle formation (Decho, 1990; Wingender et al., 1999). In fact, the idea that hydrophobic interactions might be at play in marine gel formation had been previously introduced by Stoderegger and Herndl (2004) in their observation that the gel-inducing properties of various EPS is related to their relative hydrophobicity. However objective validation of EPS on DOM assembly and the mechanism whereby EPS interacts with DOM polymers had not been investigated. Hydrophobic interactions can be qualitatively described as phenomena that induce hydrophobic species to aggregate or form clusters. However, notwithstanding more than two decades of research a formal quantitative understanding of the nature of the interaction among hydrophobic species still remains obscure. Although the source of the characteristic strong pull between hydrophobic surfaces has been well characterized, a theory to account for the multiple and often contradictory results is still missing (for review see Meyer et al., 2006). Water based cosolutes of polymer and surfactant-like amphiphilic moieties have received a great deal of attention (Lindman et al., 1993; Kuhn et al., 1998; De Gennes, 1990), and the phase behaviors of these systems can be qualitatively described within the Flory–Huggins theory (Karlström et al., 1990). However, the mechanism whereby submicellar concentrations of amphiphilic moieties like detergents can induce assemble of natural and synthetic polymers at very low concentrations is remarkably complex and not yet clearly understood (Lindman et al., 1993; Kuhn et al., 1998; Diamant and Andelman, 1999; Meyer et al., 2006). Nonetheless, the phenomenology of hydrophobic interactions remains well defined and its features resemble very much the effect of amphiphilic SEP on DOM polymer assembly. Our results show that EPS can self-assemble and that minute concentrations (300 nM) of Sagittula EPS (SEP) can readily induce assembly of DOM polymers and formation of microscopic gels (Fig. 1A, B). We conducted experiments to investigate the effect of temperature in SEP self-assembly. The enhancement of hydrophobic interactions with temperature in amphiphilic polymers has been well described in the past; it is thought to result from temperature-induced conformational changes that lead to increased hydrophobic contact area and increased probability of interchain or surface bonding (Haidacher et al., 1996). However, only empirical relationships resulting from calorimetric or chromatography studies have been published and detailed mechanisms remain obscure (Meyer et al., 2006). Although we did not conduct systematic calorimetric experiments to confirm the characteristic correlation of enthalpy and entropy described in hydrophobic interactions, we found that SEP self-assembly kinetics exhibit the characteristic nonlinear temperature dependence of hydrophobic processes with a typical increase in cooperativity that reach a Hill coefficient of ∼4.6 at 30 °C (Fig. 2). Furthermore, both SEP self-assembly in ASW and the fast SEP-induced DOM polymer assembly remain unchanged in Ca2+-free seawater (Fig. 3A, B). This outcome indicate that unlike the DOM self-assembly previously observed by Chin et al. (1998) EPS polymer assembly exhibit a faster kinetic that does not result from counterion bonding but probably from a decrease of the critical assembly concentration. The assembly of polymer-surfactant cosolutes consistently takes place at

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much lower concentration than the critical assembly concentrations of polymer or surfactant alone (Israelachvili, 1991). Moreover, in polyelectrolyte systems at high salt concentration, like the SEP/DOM system, the critical assembly concentrations becomes independent of polymer charge and remains proportional to the presence of surfactant over a large range of concentrations (Diamant and Andelman, 1999). Although most theories about polymer-surfactant assembly have been based on generalizations of micellation theory (Israelachvili, 1991), De Gennes' (1976) formulation of polymer solvation in mixed good solvents close to critical point provides a simple qualitative paradigm to understand the effect of SEP. Accordingly, changes the SEP binding could change DOM polymer flexibility inducing partial collapse of DOM polymer chains with burial of hydrophobic domains away from water and subsequent decrease of DOM critical assembly concentrations (De Gennes and Leger, 1982; Diamant and Andelman, 1999). However, interpreting our results at the light of De Gennes' (1976) theory requires objective validation of the amphiphilic properties of SEP. Although EPS are known to contain amphiphilic moieties (Decho, 1990; Passow, 2000; Bhaskar and Bhosle, 2005) and the composition of SEP strongly suggests the presence of hydrophobic domains (Alvarado Quiroz et al., 2006; Hung et al., 2005), direct evidence of functional hydrophobic properties of SEP had been missing. We tested three independent experimental lines of evidence to verify the presence of functional hydrophobic domains in SEP. First, we used Nile Red, a fluorescence probe whose fluorescence depends on the polarity of the local surrounding solvent that in the case of water is drastically affected by hydrophobic molecules. In water, Nile Red emission is negligible but increases drastically in the presence of hydrophobic moieties or hydrophobic domains within single molecules. It has been used extensively in spectroscopy as a hydrophobic probe for detection of lipids or hydrophobic domains in proteins and synthetic polymers (Hou et al., 2000; Yablon and Schilowitz, 2004). The increase of fluorescence emission intensity from Nile Red (λex = 575 nm; λem = 633 nm) upon addition of SEP implies a local decreased solvent polarity that point strongly to the existence of hydrophobic domains in SEP (Fig. 4). Second, an additional surprising and convenient finding is that SEP is itself fluorescent (λex = 285 nm, λem = 580 nm) and its emission spectrum falls within the window of excitation of Nile Red (λex = 575 nm). This feature allowed us to conduct experiments of fluorescence resonance energy transfer (FRET) to further verify that hydrophobic domains are indeed present in SEP. The protocol consists in establishing a control by exciting Nile Red (13 µM) in ASW at 285 nm that as expected produces no emission since Nile Red excitation is at 575 nm. This control is important to make sure that any 633 nm emission collected after addition of SEP results only from FRET. Addition of 25–100 µg × L− 1 SEP results in a proportional increase of Nile Red emission at λem = 633 nm (Fig. 5). This outcome shows that the 580 nm emission of SEP is exciting Nile Red, producing a characteristic 633 nm FRET emission. These results validate the idea that Nile Red is probably binding to hydrophobic domains in SEP that lay in close proximity (b100 Å) of the fluorescence emission domains of SEP. Finally, addition of 24 nm polystyrene hydrophobic beads to 0.2 µm-filtered seawater results in an quick DOM polymer assembly providing independent indica-

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tion that hydrophobic interactions, similar to those produced by SEP can indeed induce DOM assembly (Fig. 6). Although marine bacteria are probably one of the most diverse groups of micro organisms and our observations are limited only to EPS from Sagitulla, the present findings open an exciting new front of exploration to understand the microgel/bacteria interaction in the ocean. Unlike Ca-crosslinked DOM assembly that yields a huge mass of assembled DOM, EPS-induced assembly the role of bacterial EPS on global DOM assembly/dispersion equilibrium remains to be established. However, at the local scale of micro organisms, release of EPS could be an excellent strategy whereby bacteria could concentrate substrate. Release of EPS could readily induce DOM polymers to form nearby DOM networks making substrate readily accessible to bacterial ecto-enzymes. This inference is consistent with the finding that bacteria are found in “hot spots” (Azam, 1998) probably in their ESP-induced DOM networks or in colonizing DOM self-assembled networks (Moon et al., 2007). An important corollary of the present and previous work is that marine gel formation could have a dramatic effect on marine DOM cycling via the microbial loop. The basis for the paradigm that became known as the microbial loop was introduced by Pomeroy (1974) who suggested that the ocean contains a “web of consumers” in which bacteria take up DOM and inorganic nutrients and are then grazed by protozoa — who in turn are preyed upon by mucus net makers and small zooplankton that act as conduits to higher trophic levels. This inference was supported by observations of Azam and Hodson (1977) that a large fraction of heterotrophic activity in the ocean is by free-living microorganisms, presumably bacteria. The microbial loop model was formalized by Azam et al. (1983) who, based on new estimates of bacterial biomass and productivity, concluded that bacteria utilize 10–50% of all the organic carbon produced by photosynthesis. They presented evidence that DOM of ultimate phytoplankton origin is efficiently taken up by bacteria which are grazed by nanoplanktonic heterotrophic flagellates of sufficient size to pass material and energy up into the grazing food chain (Cho and Azam, 1988). However, a still unresolved critical question in microbial loop dynamics is that free-living bacteria using DOM molecules and inorganic nutrients, and motile protozoa grazing bacteria, face critical obstacles in capturing their substrate in a world of viscous (low Reynolds number) water. Within these boundary conditions, the effectiveness of DOM uptake by bacteria become fundamentally limited by low concentrations (e.g. large average distances) and restricted mobility (Fenchel, 1984; Jumars, 1993). By creating porous microenvironments of macromolecules spontaneously, continually, and reversibly assembling into concentrated networks, gels might profoundly alter these spatial constraints and drastically change the spatial and temporal dynamics of the nutrients and organisms that sustain the microbial loop (Kepkay, 1994; Kiørboe and Jackson, 2001; Moon et al., 2007). Patchiness at this fine scale could potentially facilitate, or inhibit, the dynamics of material and energy flows in the ocean, depending upon the rate at which DOM and bacteria associated with different types of gels are utilized. Considering the critical importance of the microbial loop, formation of marine gel might have consequences that scale to higher trophic levels and global element cycles (Wells, 1998).

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Y.-X. Ding et al. / Marine Chemistry 112 (2008) 11–19

In summary, the present results complement previous work on the application of theory and methods of polymer networks to investigate the molecular mechanisms of association of marine biopolymers and add to a substantial body of work on the remarkable features of bacterial EPS. Our results illustrate a unique role of these biopolymers in marine microgel formation that could be critical for the capture and concentration of microbial substrate. Our findings are limited to EPS from Sagittulla stellata; however, the specialized and active roles of microbial EPS and their interactions with DOM merit further investigations for other ecologically important microbial species. Finally, our findings substantiate the notion that there are multiple pathways of physical associations that could drive the transfer of reduced carbon from a highly diluted DOM pool to form the discrete concentrated porous networks that make seawater microgels an important link for substrate mass transfer in the World Ocean. Acknowledgments We gratefully acknowledge the chemical analysis of the EPS from Sagittula stellata by Chen Xu. This work was supported by grant 0120579 from the Biocomplexity Program of the National Science Foundation (NSF), Div. of Bioengineering and Environmental Sciences to Pedro Verdugo (PV); and NSF grants BES-0210865 and OCE-0351559 to Peter H. Santschi and Chin-Chang Hung (C-CH); Wei-Chun Chin (W-CC) and C-CH were partially supported by a grant from University of California Pacific Rim Research Program. Yong-Xue Ding (Y-XD) and Anthony Rodriguez were supported by a grant from the Alcoholic Beverage Medical Research Foundation to W-CC. This research project was conducted at PV's laboratory, University of Washington Friday Harbour Laboratories as part of Y-XD's Ph.D. dissertation at Florida State University under the mentorship of W-CC.

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