Radiolabeled Diatom Cultures
\nNatural seawater with a salinity of 35, collected from the Gulf of Mexico, was sequentially filtered through a
\n0.2 \u03bcm polycarbonate cartridge and ultrafiltered with a 1000 amu cutoff membrane to remove particulate
\nand colloidal organic matter [Guo et al., 1995; Roberts et al., 2009]. The <1000 amu ultrafiltrate fraction was
\nthen used for later experiments. The 234Th tracer was milked and purified from a 238U solution [Alvarado
\nQuiroz et al., 2006; Quigley et al., 2002]; 233Pa, in equilibrium with 237Np, was obtained from Pacific Northwest
\nNational Laboratory; 210Pb, in 1 mol L-1 nitric acid (HNO3), was purchased from Eckert & Ziegler Isotope
\nProducts, and the 7Be tracer solution (in 0.1 mol L-1 hydrochloric acid, HCl) was manufactured at the Paul
\nScherrer Institute, Switzerland [Schumann et al., 2013].
\nAutoclaved f/2 media (50 ml) were added to preconditioned clear polyethylene containers, and then ~10 to
\n15 Bq of each radionuclide tracer (234Th, 233Pa, 210Pb, and 7Be) was added. In each radiolabeled medium, 1ml
\nof laboratory axenic culture, Phaeodactylum tricornutum (UTEX 646), was then added and incubated at a temperature of 19 \u00b1 1\u00b0C with a light:dark cycle of 14 h:10 h under an irradiance of 100 \u03bcmol quanta m-2 s-1.
\nIncubation experiments were carried out in duplicate. The growth status of P. tricornutum was monitored
\nby changes in optical density at 750nm (OD750) in a parallel nonlabeled culture. When P. tricornutum reached the stationary phase observed by its OD750, cells were harvested for further extraction and analyses. The total incubation
\ntime was 35 days.
\nExopolymeric Substance (AEPS and NAEPS) Extraction
\nAEPS and NAEPS extractions were performed following the procedures described in Chuang et al. [2014, 2015]. Briefly, for NAEPS, laboratory cultures were centrifuged (2694 g, 30 min) and filtered (0.2 \u03bcm). The filtrate was desalted via diafiltration with a 1000 amu cutoff cross-flow ultrafiltration membrane, followed by freeze drying
\nfor later use. For the AEPS extraction, diatom cells were collected after centrifugation from the previous step. Then,
\nthe pellet was soaked with 0.5mol L -1\u00a0 sodium chloride (NaCl) solution for 10 min, followed by centrifugation at
\n2000 g for 15 min to remove the medium and weakly bound organic material on the cells. The pellet was
\nthen resuspended in a fresh 100 ml, 0.5mol L-1 NaCl solution and stirred gently overnight at 4\u00b0C. The resuspended
\nparticle solution was ultracentrifuged at 12,000 g (30 min, 4\u00b0C), and the supernatant was then filtered through a 0.2 \u03bcm polycarbonate membrane. The filtrate was further desalted via diafiltration with a 1000 amu cutoff ultrafiltration membrane and subsequently freeze dried for later use.\u00a0
Intracellular and Frustule BF Extraction
\nProcedures for frustule biopolymers extraction adapted from Scheffel et al. [2011]. Briefly, the
\nclean diatom cells from the previous AEPS extraction step were resuspended in 10 ml, 100mmol L-1 EDTA
\n(pH 8.0) at 4\u00b0C overnight. EDTA solution was used to extract the intracellular material after cell lysis. The supernatant
\nwas collected after centrifuging at 3000 g for 10 min, defined as EDTA extractable BF. Subsequently,
\nthe pellet was placed in 10 ml, 1% SDS in 10mmol L-1 Tris (pH 6.8) solution and heated at 95\u00b0C for 1 h.
\nThe resulting frustules were collected by centrifugation (2500 g, 10 min), washed with 10 ml milli-Q water 3
\ntimes, and then were freeze dried for later use. The supernatant from this step was collected and defined
\nas SDS extractable BF, mostly composed of soluble cell-membrane-associated materials. These two fractions
\nrepresent intracellular biopolymers lysed after cell breakage.
HF digestion was applied to help extract the diatom frustule biopolymers. HF is a nonoxidizing acid commonly
\nused to convert SiO2 to volatile SiF4 during wet digestion [Scheffel et al., 2011; \u0160ulcek and Povondra,
\n1989]. Hence, frustule biopolymers could be separated from the digested solution by a 3 kDa cutoff membrane. However, high-concentration HF would also liberate A type metal radionuclides (Th, Pa, and Be in
\nthis study) from any complex by frustule biopolymers [e.g., Burnett et al., 1997]. Furthermore, deglycosylation
\nmight also have occurred during a HF digestion [Mort and Lamport, 1977]. Therefore, the <3000 amu fraction
\nrepresents the sum of silica frustules and broken down frustule biopolymer residues.
Subsequently, 5 ml, 52% HF was added to the frustules in a 15ml plastic centrifugation tube, and the mixture
\nsolution was incubated on ice for 1 h. Hydrogen fluoride was then evaporated under an N2 stream to reduce
\nthe volume to dryness. The remaining material was neutralized with 3ml Tris\u2013HCl (250mmol L-1, pH 8.0) and
\nfollowed by centrifugation at 11,000 g for 15 min with 3000 amu Microsep centrifugal filter tubes (Milipore).
\nThe filtrate was collected and defined as the fraction of digested silica with <3000 amu frustule BF residues.
\nThe supernatant (defined as>3000amu HF soluble BF, e.g., silaffin) was concentrated to 250 \u03bcL and rinsed with
\nmilli-Q water. The pellet from this step was then washed by a 3 ml, 200mmol L-1 ammonium acetate solution
\ntwice with centrifugation at 3000 g for 20 min. The pellet was then resuspended in a 2 ml, 100mmol L-1
\nammonium acetate solution and was sonicated for 20 s until the mixture solution appeared homogenized.
\nAfter ultracentrifuging the mixture solution at 12,000 g for 5 min, the pellet (>3000 amu HF insoluble BF,
\ne.g., cingulin) was collected and freeze dried for later use. Combined BF from all three HF fractions represented
\nfrustule-embedded biopolymers.
Activity concentrations of 234Th, 233Pa, 210Pb, and 7Be were measured by counting the gamma decay energies at 63.5 keV, 312 keV, 46.5 keV, and 477.6 keV, respectively, on a Canberra ultrahigh purity germanium well detector. The 210Po activity was analyzed by liquid scintillation counting (Beckman Model 8100 Liquid Scintillation Counter).
\nConcentrations of total carbohydrate (TCHO) were determined by the TPTZ (2, 4, 6-tripyridyl-s-triazine) method using glucose as the standard and [Hung and Santschi, 2001]. Protein content was determined using a modified Lowry protein assay, using bovine serum albumin as the standard (Pierce, Thermo Scientific). uronic acids (URA) were measured by the metahydroxyphenyl method using glucuronic acid as the standard [Hung and Santschi, 2001].
Elemental contents of carbon (C) and nitrogen (N), were determined by a Perkin Elmer CHN 2400 analyzer, using cysteine (29.99% C, 11.67% N) as a standard.
Percent amount of organic fractions from diatoms that bind with radionuclide. In order to investigate the importance of biogenic silica associated biopolymers on the scavenging of radionuclides, the diatom Phaeodactylum tricornutum was incubated together with the radionuclides 234Th, 233Pa, 210Pb, and 7Be during their growth phase. Normalized affinity coefficients were determined for the radionuclides bound with different organic compound classes (i.e., proteins, total carbohydrates, uronic acids) in extracellular (nonattached and attached exopolymeric substances), intracellular (ethylene diamine tetraacetic acid and sodium dodecyl sulfate extractable), and frustule embedded biopolymeric fractions (BF). Results indicated that radionuclides were mostly concentrated in frustule BF. Among three measured organic components, Uronic acids showed the strongest affinities to all tested radionuclides. Confirmed by spectrophotometry and two-dimensional heteronuclear single quantum coherence-nuclear magnetic resonance analyses, the frustule BF were mainly composed of carboxyl-rich, aliphatic-phosphoproteins, which were likely responsible for the strong binding of many of the radionuclides. Results from this study provide evidence for selective absorption of radionuclides with different kinds of diatom-associated biopolymers acting in concert rather than as a single compound. This clearly indicates the importance of these diatom-related biopolymers, especially frustule biopolymers, in the scavenging and fractionation of radionuclides used as particle tracers in the ocean.
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BCO-DMO Processing Notes:
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\n- modified parameter names to conform with BCO-DMO naming conventions
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