The seawater (< 1 kDa) was enriched with f/2 nutrients, trace metals and vitamins, and autoclaved in pre-combusted and seawater-preconditioned clear glassware. Known activity of 59Fe (gamma emitting radionuclide) and 238Pu (alpha emitting radionuclide) were added into the seawater in pre-combusted and seawater-preconditioned clear glassware.\u00a0
\nAfter checking the pH of each radiolabeled medium to be 8.0, laboratory axenic Skeletonema costatum (UTEX LB 2308) and Emiliania huxleyi (CCMP 371) was added to 100 mL of media and incubated at a temperature of 19\u00b11\u00baC with a light:dark cycle of 14 h:10 h under an irradiation condition of 100 \u00b5mol-quanta/m2/s.
\nThe sequential chemical extraction scheme for obtaining individual fractions from S. costatum and E. huxleyi followed the procedures described in Chuang et al. (2015) and Lin et al. (2017), with a few exceptions. For the extracellular biopolymers excreted by the phytoplankton, non-attached exopolymeric substances (NAEPS) in the surrounding seawater and attached EPS (AEPS) associated with cellular surface, were harvested. Laboratory cultures were centrifuged at 3000 x g for 30 min, followed by filtration of the supernatant which was further concentrated and desalted with nanopure water (18.2 \u03a9) in 3 kDa Microsep centrifugal filter tubes (Milipore) to obtain the NAEPS fraction, while the resultant pellet from the centrifugation was resuspended by 50 mL 3% NaCl solution and stirred gently overnight at 4\u00baC to extract EPS from the cellular surface. The solution was also centrifuged, and the supernatant containing the AEPS was then filtered to remove residual cells before further desalting via the 3 kDa ultrafiltration centrifugation tubes. The final volume of concentrated solution of each biopolymer fraction (>3 kDa) was 2 mL.
\nFor the S. costatum cultures, 10 mL of 100 mM EDTA (pH 8.0) solution was added to the diatom cells from the previous AEPS extraction step. The diatom cells were resuspended at 4\u00baC overnight to extract the intracellular material after diatom cell lysis and the supernatant was collected after centrifugation to obtain the EDTA-extractable intracellular biopolymers. Then, the resultant pellet was further resuspended in 10 mL of 1% SDS/10 mM Tris (pH 6.8) solution and heated at 95\u00baC for 1 hr. The centrifuged supernatant was also collected and defined as SDS-extractable biopolymer in S. costatum cells.\u00a0
\nTo access the diatom frustule-associated biopolymers, 5 mL of 52% HF was then added to the frustules and incubated on ice for 1 hr. After the separation of HF-insoluble pellet, the HF-soluble fraction was evaporated under N2 stream and neutralized, followed by the 3 kDa centrifugal filtration to collect the digested frustule silica fraction (<3 kDa) and HF-soluble frustule-associated biopolymer (>3 kDa). Lastly, the residue biopolymer in the HF-insoluble pellet was collected with the resuspension in a 2 mL of 100 mM ammonium acetate solution and sonication. Similar to NAEPS and AEPS, all the S. costatum cellular biopolymers were concentrated and desalted with nanopure water in 3 kDa Microsep centrifugal filter tubes (Milipore).
\nThe coccosphere of the E. huxleyi cells was first dissolved before the extraction of intracellular biopolymers. In brief, the pellet from the previous AEPS extraction step was digested in 0.44 M acetic acid (HAc) (weak acidity and non-oxidizing nature to avoid the breakage of cells) plus 0.1 M NaCl solution at 4\u00baC for 8 hr. After the digestion, the mixed solution was centrifuged and filtered, followed by ultrafiltration of the supernatant with 3 kDa Microsep centrifugal filter tubes. The retentate (>3 kDa) was defined as coccosphere-associated biopolymers, and the permeate fraction (<3 kDa) was also collected to obtain the fraction of digested biogenic calcite.
\nThe E. huxleyi cells after the removal of shells were further heated in 20 mL of 1% SDS/10 mM Tris mixed solution (pH 6.8) at 95 \u00baC for 1 hr. The supernatant was also collected through centrifugation and filtration, followed by desalting with 3 kDa Microsep centrifugal filter tubes. Subsequently, the remaining pellet was further digested by 0.04 M NH2OH\u2022HCl/4.35 M HAc mixture at 96 \u00baC for 6 hr to obtain the intracellular metabolitic biopolymer. The sum of these two fractions represents the intracellular biopolymers in E. huxleyi cells.
\nAll the solutions from the different extraction steps, including the >3 kDa biopolymer fractions and the permeate (< 3 kDa, i.e., frustule and coccosphere), were counted to determine the activity of 59Fe and 238Pu. 59Fe activity was directly obtained from a Canberra ultrahigh purity germanium well gamma detector at the decay energies of 1099 kev. All the solutions for the gamma counting had the same volume and geometry to avoid geometry corrections, and all the data were decay corrected.\u00a0
\n238Pu activities were determined by alpha-spectroscopy (Xu et al., 2016). Briefly, a known activity of 242Pu was spiked to trace the yield of 238Pu during the extraction steps. The samples were oven-dried, then heated at 600 \u00baC overnight in a ceramic crucible. The resulting ash fraction was then digested in Teflon tubes overnight in concentrated HNO3 and HCl (1:1) at 85\u00baC. The remaining solid residual fraction was collected by centrifugation and discarded, and the supernatant was further evaporated to incipient dryness. To convert all Pu ions to Pu(IV), a FeSO4\u20227H2O (0.2 g/mL) solution, followed by 0.25 g of NaNO2, were added to each sample to achieve a final volume of 3 mL for each sample. Samples were then passed through an UTEVA column (Cat. # UT-C50-A, Eichrom, USA) to separate Pu from other alpha-emitting radionuclides (e.g., 238U, 241Am). After washing the column with an 8 M HNO3 solution, the Pu was eluted using freshly-prepared 0.02 M NH2OH\u2022HCl/0.02 M ascorbic acid in 2 M HNO3. The Pu-containing eluent was evaporated and re-constituted in 0.4 M (NH4)2SO4 (pH~2.6) for electroplating onto a stainless steel planchet at 0.6 Amps current for 2 hr. Sample-bearing planchets were then analyzed via alpha spectroscopy for at least one week to obtain counting errors (1 sigma) lower than 5%.
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Subsamples were taken from the concentrated biopolymers for the analysis of protein, total carbohydrate (TCHO) and uronic acid (URA), respectively. In brief, the protein abundance was measured through a modified Lowry protein assay, using bovine serum albumin (BSA) as the standard. For the concentrations of TCHO, samples were hydrolyzed by 0.09 M HCl (final concentration) at 150\u00baC for 1 h. After neutralization with NaOH solution, the hydrolysate was measured by the 2,4,6-tripyridyl-triazine (TPTZ) method (Hung et al., 2001), with glucose as the standard. URA concentrations were determined by the metahydroxyphenyl method using glucuronic acid as the standard (Hung and Santschi, 2001).\u00a0
Iron (Fe), a micronutrient for algal growth, and plutonium (Pu), an anthropogenic radionuclide, share some common features. This includes similar oceanic distributions when different input modes are taken into account, as well as their chemical behavior, such as a high affinity to natural organic matter (NOM). The NOM produced by various phytoplankton communities can potentially influence Fe cycling in the ocean, and likely also influence the transport behavior of Pu. We conducted laboratory incubation experiments using the coccolithophore Emiliania huxleyi and the diatom Skeletonema costatum, in the presence of 59Fe and 238Pu as radiotracers, in order to differentiate Fe and Pu uptake by extracellular exopolymeric substances (EPS) and intracellular biopolymers. The Fe and Pu distributions in select organic compound classes including proteins, total carbohydrates (TCHO) and uronic acids (URA) produced by these two types of phytoplankton were compared. Our results indicated that most of the Fe and Pu (>95%) were found concurrently concentrated in E. huxleyi-derived non-attached EPS, while much less (<2%) was present in the intracellular fraction of E. huxleyi. By contrast, in the diatom S. costatum, both Fe and Pu distribution was EPS > intracellular biopolymers > outer cell covering (i.e., frustule). In fact, over 50% of Fe was concentrated in S. costatum-derived attached EPS and intracellular biopolymers. The diatom derived Fe-EPS complexes were more hydrophobic, with stronger tendency to aggregate in seawater. Fe binding to biopolymers in both E. huxleyi and S. costatum cultures was related to URA concentrations, but the overall distribution of URA between these two phytoplankton species was different. Our findings suggest that the presence of URA in S. costatum cellular surface (i.e., attached EPS) and its intracellular fraction could be an indicator for the Fe transport from the surrounding seawater to the diatom cells. However, for the coccolithophore E. huxleyi, Fe appeared not to be efficiently taken up during its growth. Instead, the more hydrophilic non-attached EPS (i.e., low protein/TCHO ratio) produced by E. huxleyi could have stabilized Fe in the colloidal form as Fe-EPS complexes. Similar partitioning behavior of Fe and Pu suggests that Pu isotopes can potentially serve as a tracer for the Fe biogeochemistry in the ocean.
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BCO-DMO Processing Notes:
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\n- combined the two spreadsheets on\u00a0type and Biopolymer_fraction