Coccolithophore-associated organic biopolymers for fractionating particle-reactive radionuclides (234Th, 233Pa, 210Pb, 210Po, and 7Be) on 2018-05-15 (NCEI Accession 0291947)

Acquisition Description:
The seawater (< 1 kDa) was enriched with f/2 nutrients, trace metals and vitamins, and autoclaved in pre-combusted and seawater-preconditioned clear glassware. Then, ~50 Bq of each gamma emitting radionuclide, including 234Th, 233Pa, 210Pb and 7Be, was added. Since 210Po emits no gamma radiation, 210Po was added separately into the seawater. After checking the pH of each radiolabeled medium to be 8.0, 2 mL of laboratory axenic 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.

Non-attached exopolymeric substances (NAEPS) and exopolymeric substances attached on the coccolithophore cellular surface (AEPS) were extracted followed the procedures described in Chuang et al. (2015) and Xu et al. (2011). In brief, laboratory cultures were centrifuged at 3000 ×g for 30 min, and then the supernatant for the NAEPS fraction was filtered, followed by the concentration and extensive desalting of supernatant against nanopure water (18.2 Ω) with 3 kDa Microsep centrifugal filter tubes (Milipore). For AEPS extraction, the resultant pellet from the centrifugation was resuspended by 50 mL 3% NaCl solution and stirred gently overnight at 4ºC. Lastly, the solution was centrifuged, and the supernatant containing the AEPS was then filtered before further desalting via the 3 kDa ultrafiltration centrifugation tubes. The pellet from the previous step was thus further digested in the 0.44 M HAc + 0.1 M NaCl solution at 4ºC for 8 h. 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. The permeate (<3 kDa), defined as the fraction of digested biogenic calcite. Cells remaining from the last step was further heated in 20 mL of 1% SDS containing 10 mM Tris solution (pH 6.8) at 95 ºC for 1 h. The supernatant was also collected through centrifugation and filtration, followed by desalting and concentration with 3 kDa Microsep centrifugal filter tubes. Subsequently, the pellet was further digested by 0.04 M NH2OH HCl + 4.35 M HAc mixture at 96 ºC for 6 h, with occasional agitation to obtain the intracellular Fe-Mn associated metabolitic biopolymer. The sum of these two fractions represents the intracellular biopolymers after cell breakage.

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).

All the solutions from the different extraction steps, including the >3 kDa biopolymer fractions and the permeate (< 3 kDa), were counted for the activity concentrations of 234Th, 233Pa, 210Pb and 7Be by a Canberra ultrahigh purity germanium well gamma detector. In addition, the 210Po activity in different separately incubated fractions was determined by Beckman Model 8100 Liquid Scintillation Counter.