Abstract:
This dataset contains laboratory incubation experiments conducted 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 dataset compared 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, and includes the percentage of 59Fe and 238Pu activity and amounts of organic components in terms of µM-C, as well as the ratio of proteins to total carbohydrates (TCHO) and the percentage of the uronic acid (URA) in the bulk total carbohydrates pool, in different biopolymer fractions of E. huxleyi cells. Protein abundance was measured through a modified Lowry protein assay, using bovine serum albumin (BSA) as the standard. URA concentrations were determined by the metahydroxyphenyl method using glucuronic acid as the standard. This dataset supports the publication: Santschi, P., Quigg, A., Schwehr, K., & Xu, C. (2019). Partitioning of iron and plutonium in exopolymeric substances and intracellular biopolymers: a comparison study between the coccolithophore Emiliania huxleyi and the diatom Skeletonema costatum. Biological and Chemical Oceanography Data Management Office. doi:10.1575/1912/bco-dmo.764480.1
Methods:
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. After 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±1ºC with a light:dark cycle of 14 h:10 h under an irradiation condition of 100 µmol-quanta/m2/s.
The 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 Ω) 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 4ºC 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.
For 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 4ºC 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 95ºC for 1 hr. The centrifuged supernatant was also collected and defined as SDS-extractable biopolymer in S. costatum cells.
To 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).
The 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 4ºC 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.
The 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 ºC 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•HCl/4.35 M HAc mixture at 96 ºC for 6 hr to obtain the intracellular metabolitic biopolymer. The sum of these two fractions represents the intracellular biopolymers in E. huxleyi cells.
All 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.
238Pu 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 ºC overnight in a ceramic crucible. The resulting ash fraction was then digested in Teflon tubes overnight in concentrated HNO3 and HCl (1:1) at 85ºC. 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•7H2O (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•HCl/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%.
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 150ºC 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).
Provenance and Historical References:
Chuang, C.-Y., Santschi, P. H., Xu, C., Jiang, Y., Ho, Y.-F., Quigg, A., Guo L., Hatcher PG., Ayranov M., Schumann D. (2015). Molecular level characterization of diatom-associated biopolymers that bind234Th,233Pa,210Pb, and7Be in seawater: A case study withPhaeodactylum tricornutum. Journal of Geophysical Research: Biogeosciences, 120(9), 1858–1869. doi:10.1002/2015jg002970
Hung, C.-C., Tang, D., Warnken, K. W., & Santschi, P. H. (2001). Distributions of carbohydrates, including uronic acids, in estuarine waters of Galveston Bay. Marine Chemistry, 73(3-4), 305–318. doi:10.1016/s0304-4203(00)00114-6
Hung, C.-C., & Santschi, P. H. (2001). Spectrophotometric determination of total uronic acids in seawater using cation-exchange separation and pre-concentration by lyophilization. Analytica Chimica Acta, 427(1), 111–117. doi:10.1016/s0003-2670(00)01196-x
Lin, P., Xu, C., Zhang, S., Sun, L., Schwehr, K.A., Bretherton, L., Quigg, A., Santschi, P.H. (2017).Importance of coccolithophore-associated organic biopolymers for fractionating particle-reactive radionuclides (234 Th, 233 Pa, 210 Pb, 210 Po, and 7 Be) in the ocean. Journal of Geophysical Research: Biogeosciences, 122(8), 2033–2045. doi:10.1002/2017jg003779
Xu, C., Zhang, S., Sugiyama, Y., Ohte, N., Ho, Y.F., Fujitake, N., Kaplan D.I., Yeager C.M., Schwehr K., Santschi, P. H. (2016). Role of natural organic matter on iodine and 239,240Pu distribution and mobility in environmental samples from the northwestern Fukushima Prefecture, Japan. Journal of Environmental Radioactivity, 153, 156–166. doi:10.1016/j.jenvrad.2015.12.022
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