Partitioning of iron and plutonium to exopolymeric substances and intracellular biopolymers: A comparison study between the coccolithophore Emiliania huxleyi and the diatom Skeletonema costatum

Marine Chemistry 218 (2020) 103735

Contents lists available at ScienceDirect

Marine Chemistry journal homepage: www.elsevier.com/locate/marchem

Partitioning of iron and plutonium to exopolymeric substances and intracellular biopolymers: A comparison study between the coccolithophore Emiliania huxleyi and the diatom Skeletonema costatum

T

Peng Lina, , Chen Xua, Wei Xinga, Luni Suna, Kathleen A. Schwehra, Antonietta Quiggb,c, Peter H. Santschia,c ⁎

a b c

Department of Marine Science, Texas A & M University Galveston Campus, Galveston, TX 77553, USA Department of Marine Biology, Texas A & M University Galveston Campus, Galveston, TX 77553, USA Department of Oceanography, Texas A & M University, College Station, TX 77843, USA

ABSTRACT

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 produced by these two types of phytoplankton, including proteins, total carbohydrates (TCHO) and uronic acids (URA), were compared. Our results indicated that most of the Fe and Pu (> 95%) were found concentrated in E. huxleyi-derived non-attached EPS, while much less (< 2%) was present in the intracellular fraction of E. huxleyi. In contrast, in the diatom S. costatum, Fe and Pu were both distributed with EPS > intracellular biopolymers > outer cell covering (i.e., frustule). In fact, over 50% of the 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 (e.g., high intracellular abundance of URA in S. costatum but low intracellular URA abundance in E. huxleyi). Our findings suggest that the presence of URA on cellular surfaces of S. costatum (i.e., attached EPS) and its high intracellular fraction could be an indicator for Fe transport from the surrounding seawater to the diatom cells. However, for the coccolithophore E. huxleyi, Fe was not 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 Fe biogeochemistry in the ocean.

1. Introduction Iron (Fe) serves as an essential micronutrient element for primary productivity in the ocean (e.g., Quigg et al., 2003, 2011; Quigg, 2016), with trace concentrations of dissolved Fe (dFe, Bruland et al., 1991; Wu and Luther III, 1994; Boyé et al., 2001; Buck et al., 2015; Fitzsimmons et al., 2016) that can limit microalgal growth and productivity in up to 40% of the global oceans due to its low solubility (Boyd et al., 2007; Lam et al., 2012; Moore et al., 2013). In response to the scarcity and low solubility of Fe, the assembly of ubiquitous natural organic matter (NOM) compounds in seawater provide strong complexing ligands to create highly soluble forms of dFe, consequently making > 99% of dFe strongly bound to naturally occurring organic ligands (e.g., saccharides and siderophores) in the ocean (Rue and Bruland, 1997; Boyé et al.,

⁎

2001; Gledhill and Buck, 2012; Hassler et al., 2011a, 2015). Furthermore, these organic Fe complexes have been widely demonstrated to play a critical role in controlling the bioavailability of Fe to the microorganisms in the ocean (Boyé et al., 2005; Maldonado et al., 2005; Hassler and Schoemann, 2009; Shaked and Lis, 2012). A detailed investigation on the binding of Fe to organic matter extracted from two important phytoplankton (diatoms and coccolithophores) in the ocean will allow us to better understand the biogeochemistry of Fe. Plutonium (Pu), an anthropogenic radionuclide, has been found to show similar organic-binding behavior with Fe, such as with hydroxamate siderophores, one of the strongest Fe-binding organic compounds, which also serve as the strongest binding agents for the Pu in soil environments (Boukhalf and Crumbliss, 2002; Xu et al., 2015). In the ocean, Pu, despite having different oxidation states (predominantly

Corresponding author. E-mail address: [email protected] (P. Lin).

https://doi.org/10.1016/j.marchem.2019.103735 Received 26 March 2019; Received in revised form 6 December 2019; Accepted 9 December 2019 Available online 13 December 2019 0304-4203/ © 2019 Elsevier B.V. All rights reserved.

Marine Chemistry 218 (2020) 103735

P. Lin, et al.

as IV and V; Hirose et al., 2009, 2011) than Fe (III, II), shows similar oceanic distributions when different input modes are taken into account. For example, the transient behavior of Pu has been shown to be comparable to the steady-state distribution of Fe by using 239,240 Pu/137Cs ratios where the penetration of the more particle-reactive Pu can be corrected for vertical mixing effects using 137Cs, which is also proposed as a proxy of biogeochemical processes (Hirose et al., 2009, 2011). An investigation of the binding behavior of Fe and Pu to biopolymers will also allow us to further examine the similarity of Fe and Pu distributions and cycling in the ocean and test the validity of Pu as a proxy for biogeochemistry processes. In the ocean, in-situ production and excretion of biogenic materials (e.g., biopolymers) from phytoplankton contribute to the pool of organic matter and associated complexing ligands for dFe (Aluwihare et al., 1997; Rijkenberg et al., 2008; Benner, 2011). Biopolymers can have different compositions based on the abundance of specific organic compounds (e.g., protein, acid polysaccharides and neutral sugars). For example, diatoms and coccolithophores, two of the most important contributors to global oceanic primary production (Nelson et al., 1995; Armbrust, 2009), have been found to produce biopolymers with different composition in their extracellular substances and intracellular fractions (Quigg et al., 2016). This phenomenon is responsible for different concentrations of natural radionuclides (e.g., thorium that has similar chemical properties to Fe due to similar ionic potentials, Chuang et al., 2013) in various EPS and other intracellular biopolymer fractions (Chuang et al., 2014, 2015a; Lin et al., 2017a). Accordingly, similarities or differences in the binding of Fe (and Pu) to EPS and other biopolymers produced by diatoms and coccolithophores in the ocean will be of great interest. Furthermore, Fe uptake by phytoplankton is related to the biological pump (e.g., residence time and scavenging from the euphotic layer), and could suggest differences between diatoms- and coccolithophores-dominated marine systems. To our knowledge, studies examining the potential differences in Fe (and Pu) binding with various biopolymer fractions in diatoms and coccolithophores during their growth, and how the chemical composition of biologically produced EPS and intracellular biopolymers affect that binding are still scarce. To fill this knowledge gap, cultures of the coccolithophore Emiliania huxleyi and the diatom Skeletonema costatum were incubated in the presence of 59Fe and 238Pu to examine the association of these two elements with intracellular and extracellular biopolymers produced during cell growth. This includes attached EPS (APES) and non-attached EPS (NAEPS) as well as the frustule/coccosphere. By combining these results, we assessed the relationships between Fe and Pu and the organic composition of various biopolymers in order to learn how such associations affect the cycling of Fe and Pu in the upper ocean.

radiolabeled control culture for each species was set up to monitor the pH and growth status, measured as the change in optical density at 750 nm wavelength (OD750) with a UV–Vis spectrophotometer. Once the OD750 value was constant, i.e., the stationary phase for growth, all cultures were harvested. Biopolymers were also sequentially extracted from this control culture for the measurement of the biopolymer components (See details below). 2.2. Extracellular biopolymer extraction The sequential chemical extraction scheme for obtaining biopolymer fractions from S. costatum (Fig. S1) and E. huxleyi (Fig. S2) followed the procedures described in Chuang et al. (2015a) and Lin et al. (2017a), 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 the cellular surface, were harvested. Laboratory cultures were centrifuged at 3000 xg 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 in 50 mL 0.5 M NaCl solution and stirred gently overnight at 4 °C 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. 2.3. Extraction of intracellular biopolymers and its frustule-related biopolymers from Skeletonema costatum Because of the different shell composition (silica for the diatom S. costatum and biogenic calcite for the coccolithophore E. huxleyi), different extraction procedures were applied to each species to access the intracellular biopolymer and shell-associated biopolymers (Fig. S1 and Fig. S2). 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 4 °C 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 35 mM SDS/10 mM Tris (pH 6.8) solution and heated at 95 °C for 1 h. 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 concentrated HF was then added to the frustules and incubated on ice for 1 h. After the separation of HF-insoluble pellet, the HF-soluble fraction was evaporated under N2 stream and neutralized, followed by 3 kDa centrifugal filtration to collect the digested frustule silica fraction (< 3 kDa) and the HF-soluble frustule-associated biopolymer (> 3 kDa). Lastly, the residual biopolymer in the HF-insoluble pellet was collected with resuspension in 2 mL of 100 mM ammonium acetate solution and sonication. Similar to NAEPS and AEPS, all the S. costatum biopolymer fractions were concentrated and desalted with nanopure water in 3 kDa Microsep centrifugal filter tubes (Milipore).

2. Materials and methods 2.1. Radiolabeled Skeletonema costatum and Emiliania huxleyi culturing Filtered natural seawater (pH = 8.0) collected from the Gulf of Mexico (Salinity = 35), containing only low molecular weight organic matter (< 1 kDa), was used as the culturing medium for Skeletonema costatum (UTEX LB 2308) and Emiliania huxleyi (CCMP 371). The seawater was filtered through a 0.2 μm polycarbonate cartridge to remove all particles first, followed by cross-flow ultrafiltration using a 1 kDa cutoff membrane to remove colloidal organic matter. After enrichment with f/2 medium nutrients, trace metals and vitamins, and sterilization, known activity of 59Fe (gamma emitting radionuclide) and 238Pu (alpha emitting radionuclide), both at pM level, were added into the seawater in pre-combusted and seawater-preconditioned clear glassware. Laboratory axenic S. costatum and E. huxleyi cultures were added to 100 mL of medium. All the radiolabeled incubations were conducted in duplicates at 19 ± 1 °C with a light:dark cycle of 14 h:10 h under an irradiation condition of 100 μmol-quanta/m2/s. Additionally, a non-

2.4. Emiliania huxleyi coccosphere (biogenic calcite) and biopolymer extraction 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 4 °C for 8 h. After the digestion, the mixed solution was centrifuged and filtered, followed by ultrafiltration of the 2

Marine Chemistry 218 (2020) 103735

P. Lin, et al.

Table 1 Percentage of 59Fe and 238Pu activity and amounts of organic components in terms of μM-C, 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 S. costatum cells. Activity percentage (%)

59

Fe Pu

238

Organic components Amount (μM-C) Protein TCHO URA Protein-C/TCHO-C %URA/TCHO

Extracellular biopolymer

Frustule

Intracellular biopolymer

NAEPS

AEPS

Silica frustule

Frustule-related biopolymer

SDS-extractable

EDTA-extractable

41.6 ± 14.4 33.8 ± 1.3

31.0 ± 7.1 14.8 ± 1.3

4.1 ± 0.1 6.2 ± 1.0

bdl 12.1 ± 8.1

0.8 ± 0.1 10.1 ± 2.9

22.5 ± 1.0 23.0 ± 1.8

115 17.7 7.3 6.5 41

42.1 5.4 2.4 7.8 45

na na na na na

6.8 17.1 2.9 0.4 17

95.8 17.1 3.3 5.6 19

133 38.2 12.6 3.5 33

“bdl” denotes that 59Fe or 238Pu is not detectable. “na” denotes that the sample is not measured.

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. After removal of the shells, the E. huxleyi cells were further heated in 20 mL of 35 mM SDS/10 mM Tris mixed solution (pH 6.8) at 95 °C for 1 h. 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 °C for 6 h to obtain the intracellular biopolymers. The sum of these two fractions represents the intracellular biopolymers in E. huxleyi cells. 2.5. Determination of

59

Fe and

238

in the various biopolymer fractions. This approach had been previously verified (Quigley et al., 2001). 2.6. Measurement of biopolymer components Subsamples were taken from the concentrated biopolymers for the analysis of protein, total carbohydrate (TCHO) and uronic acid (URA). These three organic compound classes were selected for the analysis as they are major organic components of the EPS and biopolymers produced by marine phytoplankton (e.g., Zhang et al., 2008). 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 °C for 1 h. After neutralization with NaOH solution, the hydrolysate was measured by the 2,4,6-tripyridyl-triazine method (Hung et al., 2001; Lin and Guo, 2015), with glucose as the standard. URA concentrations were determined by the metahydroxyphenyl method using glucuronic acid as the standard (Hung and Santschi, 2001). The measurements for all of the components was carried out in duplicates. It should be noted that TCHO also includes URA, thus the percentage of URA in the TCHO pool was estimated in the present study. Data on concentrations of protein, TCHO and URA for the E. huxleyi culture have been published elsewhere (Lin et al., 2017a), but are considered here for the discussion.

Pu activity

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 59 Fe and 238Pu. 59Fe activity was directly obtained from a Canberra ultrahigh purity germanium well gamma detector at the decay energy 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. 238 Pu activities were determined by alpha-spectroscopy (Xu et al., 2016; Lin et al., 2017b). 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 °C 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 °C. 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 alphaemitting 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 h. Sample-bearing planchets were then analyzed via alpha spectroscopy for at least one week to obtain counting errors (1 sigma) lower than 5%. The mass balance of 59Fe and 238Pu during the incubation and extraction was monitored, basically showing 85% of 59Fe and 92% of 238 Pu to be recovered. The radionuclide which was lost onto the lab ware in the present work was considered to not participate in the experiment and was excluded from the calculation of activity percentage

3. Results and discussion 3.1. Distributions of Fe and Pu in two phytoplankton cultures The activity percentage and partitioning of 59Fe and 238Pu among biopolymer fractions extracted from S. costatum and E. huxleyi are summarized in Tables 1 and 2, respectively. Distributions of 59Fe and 238 Pu in S. costatum's frustule and E. huxleyi's coccosphere are shown in Figs. 1 and 2, respectively. Considerable amounts of 59Fe and 238Pu were taken up by S. costatum cells, showing 23.3 ± 1.3% of 59Fe and 33.1 ± 3.4% of 238Pu distributed in the intracellular biopolymer fraction (sum of EDTA- and SDS-extractable intracellular biopolymers, Fig. 1; Table 1). Only 4.1 ± 0.1% of 59Fe on average was found in the silica frustule fractions, with undetectable activity in its associated biopolymers, while the 238Pu had higher concentrations in the silica frustule-associated biopolymers (12.1 ± 8.1%) than that in the silica frustule (6.2 ± 1.0%) (Fig. 1). For the extracellular EPS excreted by S. costatum cells, the NAEPS acted as the strongest binding agent for 59Fe (41.6 ± 14.4%) and 238Pu (33.8 ± 1.3%) among all the extracted biopolymer fractions, and the APES fractions accumulated more 59Fe than 238Pu (31.0 ± 7.1% vs. 14.8 ± 1.3%, Fig. 1, Table 1). In comparison, almost all the 59Fe (97.1 ± 11.2%) and 238Pu (97.9 ± 6.8%) were concentrated in the NAEPS fraction of the E. huxleyi culture 3

Marine Chemistry 218 (2020) 103735

P. Lin, et al.

Table 2 Percentage of 59Fe and 238Pu activity and amounts of organic components in terms of μM-C, 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. Activity percentage (%)

59

Fe Pu Organic componentsa Amount (μM-C) Protein TCHO URA Protein-C/TCHO-C %URA/TCHO 238

Extracellular biopolymer

Coccosphere

Intracellular biopolymer

NAEPS

AEPS

Biogenic calcite

Coccosphere-related biopolymer

SDS-extractable

Metabolitic biopolymer

97.1 ± 11.2 97.9 ± 6.8

1.1 ± 0.1 1.2 ± 0.1

0.2 ± 0.0 bdl

0.1 ± 0.0 bdl

1.5 ± 0.5 0.5 ± 0.0

bdl 0.1 ± 0.0

23.7 25.7 38.2 0.9 100

34.4 5.8 1.3 5.9 22

na na na na na

bdl 1.3 0.2 0 13

54.7 12.6 3 4.3 24

6.2 1 0.3 6.2 28

“bdl” denotes that 59Fe or 238Pu is not detectable and that protein concentration is lower than the detection limit (0.5 μM-C). “na” denotes that the sample is not measured. a Data from Lin et al. (2017a).

(Fig. 2), with minor distributions in APES (~1% for both elements) and intracellular biopolymers (1.5 ± 0.5% for 59Fe and 0.5% for 238Pu) of E. huxleyi cells (Fig. 2). Both 59Fe and 238Pu were almost undetectable in the other fractions (< 0.5%, Table 2), including the coccosphere and its associated biopolymers. The sequence of enrichment of 59Fe and 238 Pu in the different biopolymer fractions in both S. costatum and E. huxleyi cultures was similar (Figs. 1 and 2), although the two metals did not follow each other exactly in the S. costatum (e.g., minor 59Fe but ~18% of 238Pu in silica frustule and its associated biopolymers, Table 1). However, diatom-associated Fe and Pu both generally followed the order of EPS (sum of NAEPS and AEPS) > intracellular biopolymers > shell (i.e., frustule and its associated biopolymers, Table 1). Diatoms and coccolithophores ubiquitously exist in marine environments and the uptake of micronutrients (e.g., Fe) is important for the growth and productivity of phytoplankton (Coale et al., 1996; Hutchins and Boyd, 2016; Quigg, 2016). Our results provide evidence that the Pu isotopes (e.g., 239Pu and 240Pu) can generally follow the transport behavior of Fe in the ocean. Fe as a micronutrient is required by the photosynthesis system of these two types of phytoplankton (Quigg et al., 2003, 2011). Pu, though not a micronutrient required by their metabolic or biosynthetic activities, might be recognized as Fe, depending on the functions of Fe, by some biopolymers due to their similar ionic potentials. This similarity might be responsible for the observed Fe-like or nutrient-like vertical oceanic profile for Pu isotopes (after correction for conservative element 137Cs, using 239,240Pu/137Cs

ratios), for example, in the North Pacific Ocean (Boyd and Ellwood, 2010; Hirose et al., 2009, 2011). Additionally, this also provides evidence for the possibility of the Pu isotopes serving as an indicator of biogeochemical processes, especially associated with the biogeochemical cycling of the Fe (“biological iron pump”) in the upper ocean (e.g., euphotic layer). Nevertheless, more laboratory studies and field experiments are needed to test the validity of our observations, since other particles (e.g., lithogenic particles), in addition to the phytoplankton-derived biopolymers and biogenic particles, can affect the scavenging of Pu isotopes in the water column. In addition, there was also different Pu partitioning between S. costatum and E. huxleyi cultures (Fig. 1 vs. Fig. 2), with over 97% of Pu binding with EPS in the colloidal fraction of the cocolithophore culture but < 35% of Pu being stabilized as colloidal Pu-EPS complexes in the diatom culture. This suggests that if anthropogenic Pu is accidently released into the marine environment (e.g., Fukushima Daiichi Nuclear plants accident, Zheng et al., 2013; Xu et al., 2016), the diatom community may contribute more to the scavenging of Pu isotopes (> 60% of Pu distributed in diatom cellular surface, frustule and intracellular biopolymers, Table 1). In addition, as diatoms are at the base of the food chain, the Pu isotopes could be also spread through the food chain to other marine organisms by biotransference or biomagnification as has been observed for many other elements (e.g., Quigg, 2016). In contrast, anthropogenic Pu isotopes could be retained to a greater extent in the water column due to the stabilization of Pu by coccolithophore-produced colloidal EPS, which may increase the residence time Fig. 1. Percentages of 59Fe (left) and 238Pu (right) activities in different biopolymer fractions of S. costatum cells, including S. costatum-derived non-attached exopolymeric substances (NAEPS) in the surrounding seawater, S. costatum-derived exopolymeric substances attached on the cellular surface (AEPS), S. costatum intracellular biopolymers (sum of SDS- and EDTA-extractable intracellular biopolymers), silica frustule and its associated biopolymers.

4

Marine Chemistry 218 (2020) 103735

P. Lin, et al.

Fig. 2. Percentages of 59Fe (left pie) and 238Pu (right pie) activities in different biopolymer fractions of E. huxleyi cells, including E. huxleyi-derived non-attached exopolymeric substances (NAEPS) in the surrounding seawater, E. huxleyi-derived exopolymeric substances attached on the cellular surface (AEPS), E. huxleyi intracellular biopolymers (sum of SDS-extractable and metabolite intracellular biopolymer), coccosphere shell and its associated biopolymers.

of anthropogenic Pu isotopes, and might cause long-term radiation risk to the ecological health in marine environments.

difference in %URA/TCHO between diatom-derived biopolymer fractions. As shown in Table 1 and Fig. 3, the URA in AEPS on the diatom cellular surface comprised almost 45% of the bulk TCHO pool, which was comparable to the %URA/TCHO value in the NAEPS (41%) and somewhat higher than that in the EDTA-extractable intracellular biopolymers (33%). For other biopolymer fractions, the percentage of URA in the bulk TCHO pool was < 20%, showing 17% and 19% for frustuleassociated biopolymers and SDS-extractable intracellular biopolymers. Together with the 59Fe or 238Pu partitioning (Fig. 1), the enhanced binding of 59Fe and 238Pu in in the S. costatum culture evidently followed the enrichment of URA in the bulk TCHO pool (Fig. 3), in the order of NAEPS > AEPS > EDTA-extractable intracellular biopolymers > > SDS-extractable intracellular biopolymers and frustule-associated biopolymers, although the %URA/TCHO values between two

3.2. Organic binding between Fe (and Pu) and diatom/coccolithophorederived biopolymers By comparing the partitioning of 59Fe or 238Pu in the S. costatum and E. huxleyi cultures (Fig. 1 vs. Fig. 2), we found that the partitioning of 59 Fe or 238Pu between the diatom and coccolithophore is different, corresponding with the difference in abundance and composition of the organic components in various biopolymer fractions between the two cultures (Fig. 3 versus Fig. 4). For example, the partitioning pattern of 59 Fe and 238Pu in the S. costatum culture (Fig. 1) was more complicated than in E. huxleyi cultures (Fig. 2), which may be due to the smaller

Fig. 3. Percentage of organic component amounts, including proteins, total carbohydrates (TCHO) and uronic acids (URA), as well as the ratio of protein to total carbohydrate (Protein-C/TCHO-C) and the percentage of uronic acid in the total carbohydrate pool (%URA/TCHO) in each biopolymer fraction of S. costatum cells. Both the percentage and ratio were calculated in terms of μM-C. The sum of percentages of each organic component in extracellular, frustule, and intracellular biopolymers represents 100%.

5

Marine Chemistry 218 (2020) 103735

P. Lin, et al.

Fig. 4. Percentage of organic component amounts, including proteins, total carbohydrates (TCHO) and uronic acids (URA), as well as the ratio of protein to total carbohydrate (Protein-C/TCHO-C) and the percentage of uronic acid in the total carbohydrate pool (%URA/TCHO) in each biopolymer fraction of E. huxleyi cells. Both the percentage and ratio were calculated in terms of μM-C. The sum of percentages of each organic component in extracellular, coccosphere, and intracellular biopolymers represents 100%.

extracellular biopolymers was minor (Table 1). In comparison, the strong enrichment of 59Fe (97.1 ± 11.2%) and 238 Pu (97.9 ± 6.8%) in the NAEPS of the E. huxleyi culture appeared to follow the high abundance of URA in the bulk TCHO pool (i.e., %URA/ TCHO ~ 100%, Fig. 4) of the NAEPS (Table 2), which also showed the lowest relative hydrophobicity according to the ratio of protein to TCHO normalized to organic carbon (Protein-C/TCHO-C, an indicator of relative hydrophobicity of organic matter, Xu et al., 2011). This demonstrates that the hydrophilic acid polysaccharide-enriched colloidal organic matter excreted from the growing coccolithophores can strongly bind Fe (and Pu). Therefore, our laboratory results from two different phytoplankton species consistently indicate the tight association between Fe (and Pu) and URA (i.e., acid polysaccharide) in seawater. With respect to Fe, this finding was not only consistent with the previous observations showing the stabilization of dissolved Fe by the anionic polysaccharides and carboxyl groups in seawater (Nagy et al., 2003; Sreeram et al., 2004; Hassler et al., 2011b), but also provides additional evidence demonstrating that Fe stabilization by acid polysaccharides can also happen in phytoplankton intracellular biopolymers, as is observed here by the enrichment of 59Fe in EDTA-extractable intracellular biopolymers of S. costatum cells (Table 1, Fig. 1). Nevertheless, such stabilization of Fe in intracellular biopolymers was not found in the E. huxleyi cells (only 1.5 ± 0.5% for 59Fe and 0.5% for 238 Pu, Fig. 2). The high concentrations of URA in S. costatum cells (> 50% of URA distributed in intracellular biopolymers) and its AEPS (20% of URA, Fig. 3) might act as a “bridge” to facilitate the transport of dissolved Fe from the surrounding seawater into diatoms. The low abundance of intracellular URA in E. huxleyi suggests that this “bridge” may not exist for the coccolithophore community. Such a “URA-bridge” across the seawater-cellular surface-intracellular interface (Sutak et al., 2012) may be responsible for our observation that diatoms can take up

Fe from the culture medium more efficiently, compared with coccolithophores. Another possible explanation for the higher Fe-uptake efficiency of S. costatum cells might be due to the much higher abundance of proteins (which are known to be more hydrophobic, Schwehr et al., 2018; Sun et al., 2019) in S. costatum-derived biopolymers, especially in the intracellular fractions (229 μM-C for S. costatum vs. 61 μM-C for E. huxleyi, Tables 1 and 2), thus promoting the formation of Fe-protein complexes (e.g., ferritin) and enhancing Fe storage in phytoplankton (Marchetti et al., 2009; Botebol et al., 2015). Other species of diatoms and coccolithophores may have different distributions of organic components in their extracellular and intracellular biopolymers (e.g., diatom Phaeodactylum tricornutum vs. diatom Skeletonema costatum as observed in Chuang et al., 2015b), and should be considered in future studies. 3.3. Implication to Fe cycling in the ocean Fe has been widely demonstrated to limit primary productivity in up to 40% of the global ocean (Boyd et al., 2007; Hassler et al., 2011b; Marchetti and Maldonado, 2016). It is usually present at sub-nanomolar levels and strongly bound to organic ligands (Bruland et al., 1991; Boyé et al., 2001; De Jong et al., 2008). The composition of NOM and its interaction with Fe (such as Fe-EPS complexes) can play a significant role in Fe bioavailability and residence time in the ocean (Özturk et al., 2004; Morel et al., 2008; Hassler et al., 2011b). Our laboratory experiments using two different phytoplankton types showed these species produced EPS with different compositions (Fig. 3 vs. Fig. 4), accompanied with distinct partitioning behavior of Fe (Fig. 1 vs. Fig. 2). For example, NAEPS from the diatom culture appeared to have relatively higher hydrophobicity, according to the observed higher proteinC/TCHO-C values of 6.5 (Fig. 3), compared with coccolithophore-produced NAEPS (0.9, Fig. 4), consistent with the previous field or 6

Marine Chemistry 218 (2020) 103735

P. Lin, et al.

laboratory experiments demonstrating that the nature of the phytoplankton community effects the quality of EPS that are biologically produced (Hung et al., 2003; Becquevort et al., 2007). The highly hydrophilic NAEPS produced by the coccolithophores stabilized Fe in the colloidal fraction. This should result in significantly longer residence time for Fe in the upper ocean when coccolithophores are dominant. Although the coccolithophores themselves do not take up Fe efficiently (< 2% of Fe partitioned in coccolithophore intracellular biopolymers, Fig. 2), it may efficiently stabilize the Fe as a colloidal Fe-EPS complexed form that can be highly bioavailable (Hassler et al., 2011b, 2015) to other microorganisms. Colloidal Fe-EPS also has the potential to be released from sinking particles, thus accounting for Fe regeneration at depth. In contrast, in diatom-dominated areas, the relatively more hydrophobic NAEPS-Fe and AEPS-Fe complexes (Table 1) may be more readily aggregated to form marine snow (Xu et al., 2018), resulting in shorter Fe residence times. As a result, in areas dominated by diatoms (e.g., the Southern Ocean, nutrient-rich coastal waters, and during the spring bloom) the Fe biological pump may be enhanced (i.e., more dynamic) compared to areas where coccolithophores are dominant (e.g., temperate subtropical and tropical waters). Additionally, in areas where both species co-exist, the EPS-Fe complexes produced by coccolithophores may increase Fe availability to diatoms. Our results demonstrate that different phytoplankton communities produce EPS and biopolymers with different composition and physicochemical behavior (e.g., hydrophobicity, acid polysaccharide abundance), and this may influence the stabilization of Fe in the water column and the availability of Fe to the phytoplankton community.

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.marchem.2019.103735. References Aluwihare, L.I., Repeta, D.J., Chen, R.F., 1997. A major biopolymeric component to dissolved organic carbon in surface seawater. Nature 387, 166–169. Armbrust, E.V., 2009. The life of diatoms in the world’s oceans. Nature 459, 185–192. https://doi.org/10.1038/nature08057. Becquevort, S., Lancelot, C., Schoemann, V., 2007. The role of Fe in the bacterial degradation of organic matter derived from Phaeocystis antarctica. Biogeochemistry 83, 119–135. Benner, R., 2011. Loose ligands and available iron in the ocean. Proc. Nat. Acad. Sci. U. S. A. 108, 893–894. Botebol, H., et al., 2015. Central role for ferritin in the day/night regulation of iron homeostasis in marine phytoplankton. PNAS 112 (47), 14652–14657. Boukhalf, H., Crumbliss, A.L., 2002. Chemical aspects of siderophore mediated iron transport. Biometals 15 (4), 325–339. Boyd, P.W., Ellwood, M.J., 2010. The biogeochemical cycle of iron in the ocean. Nat. Geosci. 3, 675–682. Boyd, P.W., et al., 2007. Mesoscale iron enrichment experiments 1993–2005: synthesis and future directions. Science 315, 612–617. Boyé, M., van den Berg, C.M.G., De Jong, J.T.M., Leach, H., Croot, P., De Baar, H.J.W., 2001. Organic complexation of iron in the Southern Ocean. Deep Sea Res. I 48, 1477–1497. Boyé, M., Nishioka, J., Croot, P.L., Laan, P., Timmermans, K.R., De Baar, H.J.W., 2005. Major deviations of iron complexation during 22 days of a mesoscale iron enrichment in the open Southern Ocean. Mar. Chem. 96, 257–271. Bruland, K.W., Donat, J.R., Hutchins, D.A., 1991. Interactive influences of bioactive trace metals on biological production in oceanic waters. Limnol. Oceanogr. 36 (8), 1555–1577. Buck, K.N., Sohst, B., Sedwick, P.N., 2015. The organic complexation of dissolved iron along the U.S. GEOTRACES (GA03) North Atlantic section. Deep-Sea Res. II Top. Stud. Oceanogr. 116, 152–165. Chuang, C.-Y., Santschi, P.H., Ho, Y.-F., Conte, M.H., Guo, L., Schumann, D., Ayranov, m., Li, Y.-H., 2013. Role of biopolymers as major carrier phases of Th, Pa, Pb, Po, and Be radionuclides in settling particles from the Atlantic Ocean. Mar. Chem. 157, 131–143. Chuang, C.-Y., Santschi, P.H., Jiang, Y., Ho, Y.-F., Quigg, A., Guo, L., Ayranov, M., Schumann, D., 2014. Important role of biomolecules from diatoms in the scavenging of particle-reactive radionuclides of thorium, protactinium, lead, polonium and beryllium in the ocean: a case study with Phaeodactylum tricornutum. Limnol. Oceanogr. 59 (4), 1256–1266. https://doi.org/10.4319/lo.2014.59.4.1256. Chuang, C.-Y., Santschi, P.H., Xu, C., Jiang, Y., Ho, Y.-F., Quigg, A., Guo, L., Hatcher, P.G., Ayranov, M., Schumann, D., 2015a. Molecular level characterization of diatom-associated biopolymers that bind 234Th, 233Pa, 210Pb, and 7Be in seawater: a case study with Phaeodactylum tricornutum. J. Geophy. Res.: Biogeosci. 120. https://doi.org/ 10.1002/2015JG002970. Chuang, C.-Y., Santschi, P.H., Wen, L.-S., Guo, L., Xu, C., Zhang, S., Jiang, Y., Ho, Y.-F., Schwehr, K.A., Quigg, A., Hung, C.-C., Ayranov, M., Schumann, D., 2015b. Binding of Th, Pa, Pb, Po and Be radionuclides to marine colloidal macromolecular organic matter. Mar. Chem. 173, 320–329. Coale, K.H., et al., 1996. A massive phytoplankton bloom induced by an ecosystem-scale iron fertilization experiment in the equatorial Pacific Ocean. Nature 383, 495–501. De Jong, J., Schoemann, V., Mattielli, N., Lannuzel, D., 2008. High-accuracy determination of iron in seawater by isotope dilution multiple collector inductively coupled plasma mass spectrometry (ID-MC-ICP-MS) using nitrilotriacetic acid chelating resin for preconcentration and matrix separation. Anal. Chim. Acta 623, 126–139. https:// doi.org/10.1016/j.aca.2008.06.013. Fitzsimmons, J.N., Conway, T.M., Lee, J.-M., Kayser, R., Thyng, K.M., John, S.G., Boyle, E.A., 2016. Dissolved iron and iron isotopes in the southeastern Pacific Ocean. Glob. Biogeochem. Cycles 30 (10), 1372–1395. Gledhill, M., Buck, K.N., 2012. The organic complexation of iron in the marine environment: a review. Front. Microbiol. 28 (3), 1–17. https://doi.org/10.3389/fmicb. 2012.00069. Hassler, C.S., Schoemann, V., 2009. Bioavailability of organically bound Fe to model phytoplankton of the Southern Ocean. Biogeosciences 6, 2281–2296. Hassler, C.S., Schoemann, V., Nichols, C.M., Butler, E.C.V., Boyd, P.W., 2011a. Saccharides enhance iron bioavailability to Southern Ocean phytoplankton. PNAS 108 (3), 1076–1081. Hassler, C.S., Alasonati, E., Mancuso Nichols, C.A., Slaveykova, V.I., 2011b. Exopolysaccharides produced by bacteria isolated from the pelagic Southern Ocean – role in Fe binding, chemical reactivity, and bioavailability. Mar. Chem. 123, 88–98. Hassler, C.S., Norman, L., Mancuso Nichols, C.A., Clementson, L.A., Robinson, C., Schoemann, V., Watson, R.J., Doblin, M.A., 2015. Iron associated with exopolymeric substances is highly bioavailable to oceanic phytoplankton. Mar. Chem. 173, 136–147. Hirose, K., Aoyama, M., Povinec, P.P., 2009. 239,240Pu/137Cs ratios in the water column of the north Pacifc: a proxy of biogeochemical processes. J. Environ. Radioact. 100, 258–262. Hirose, K., Kim, C.S., Yim, S.A., Aoyama, M., Fukasawa, M., Komura, K., Povinec, P.P., Sanchez-Cabeza, J.A., 2011. Vertical profiles of plutonium in the central South Pacific. Prog. Oceanogr. 89, 101–107.

4. Conclusions By fractionating cultures of S. costatum and E. huxleyi that were incubated with 59Fe/238Pu, we demonstrated that the Pu isotopes in the ocean may be enriched in phytoplankton-derived biopolymers similarly to Fe. This might explain the observed Fe-like or nutrient-like vertical profile for Pu isotopes in the ocean, and also provides evidence for the possibility of Pu isotopes serving as proxies for Fe cycling in the upper ocean. Additionally, it was also found that the details of the partitioning of 59Fe or 238Pu between the diatom S. costatum and coccolithophore E. huxleyi are different, corresponding to the relative abundance and composition of the organic components in various biopolymer fractions between the two cultures. With respect to Fe, the coccolithophore E. huxleyi appeared not to efficiently take up Fe during its growth, but excreted URA-enriched and hydrophilic non-attached EPS (i.e., low protein/TCHO ratio) that stabilized > 95% of Fe in the colloidal form as Fe-EPS complexes. In comparison, a more even distribution of URA and a higher abundance of protein in diatom S. costatum-derived biopolymers may enhance the Fe uptake efficiency for this species. Significant differences in the hydrophobicity of EPS produced by diatoms vs. coccolithophores may also influence the intensity of the Fe biological pump between diatom- vs. coccolithophore-dominated waters. Acknowledgment We thank Jessica Hillhouse at Texas A&M University Galveston Campus for her assistance during phytoplankton incubation. We appreciate all the constructive comments from the Associate Editor, Dr. William Landing, and Reviewers and their time spent on our manuscript. This research was partially supported by a grant from The Gulf of Mexico Research Initiative to support consortium research entitled ADDOMEx (Aggregation and Degradation of Dispersants and Oil by Microbial Exopolymers) and ADDOMEx-2 Consortiums. This research was also partially supported by a grant from NSF (OCE#1356453). The authors cite no conflict of interest. 7

Marine Chemistry 218 (2020) 103735

P. Lin, et al. Hung, C.-C., Santschi, P.H., 2001. Spectrophotometric determination of total uronic acids in seawater using cation-exchange separation and pre-concentration by lyophilization. Anal. Chim. Acta 427 (1), 111–117. Hung, C.-C., Tang, D., Warnken, K.W., Santschi, P.H., 2001. Distributions of carbohydrates, including uronic acids, in estuarine waters of Galveston Bay. Mar. Chem. 73, 305–318. Hung, C.-C., Guo, L., Schultz, G.E., Pinckney, J.L., Santschi, P.H., 2003. Production and flux of carbohydrate species in the Gulf of Mexico. Glob. Biogeochem. Cycles 17, 1055. https://doi.org/10.1029/2002GB001988. Hutchins, D.A., Boyd, P.W., 2016. Marine phytoplankton and the changing ocean iron cycle. Nat. Clim. Chang. 6, 1072–1079. Lam, P.J., Ohnemus, D.C., Marcus, M.A., 2012. The speciation of marine particulate iron adjacent to active and passive continental margins. Geochim. Cosmochim. Acta 89, 108–124. Lin, P., Guo, L., 2015. Spatial and vertical variability of dissolved carbohydrate species in the northern Gulf of Mexico following the Deepwater horizon oil spill, 2010-2011. Mar. Chem. 174, 13–25. Lin, P., Xu, C., Zhang, S., Sun, L., Schwehr, K.A., Bretherton, L., Quigg, A., Santschi, P.H., 2017a. Importance of coccolithophore-associated organic biopolymers for fractionating particle-reactive radionuclides (234Th, 233Pa, 210Pb, 210Po, and 7Be) in the ocean. J. Geophy. Res.: Biogeosci. 120. https://doi.org/10.1002/2017JG003779. Lin, P., Xu, C., Zhang, S., Fujitake, N., Kaplan, K.I., Yeager, C.M., Sugiyama, Y., Schwehr, K.A., Santschi, P.H., 2017b. Plutonium partitioning behavior to humic acids from widely varying soils is related to carboxyl-containing organic compounds. Environ. Sci. Technol. 51, 11742–11751. Maldonado, M.T., Strzepek, R.F., Sander, S., Boyd, P.W., 2005. Acquisition of Fe bound to strong organic complexes, with different Fe binding groups and photochemical reactivities, by plankton communities in Fe-limited subantarctic waters. Glob. Biogeochem. Cycles 19https://doi.org/10.1029/2005GB002481. GB4S23. Marchetti, A., Maldonado, M.T., 2016. Iron. Devel. Devel Appl Phycol 6, 233–279. Marchetti, A., Parker, M.S., Moccia, L.P., Lin, E.O., Arrieta, A.L., Ribalet, F., Murphy, M.E.P., Maldonado, M.T., Armbrust, E.V., 2009. Ferritin is used for iron storage in bloom-forming marine pennate diatoms. Nature 457, 467–470. Moore, C.M., et al., 2013. Processes and patterns of oceanic nutrient limitation. Nat. Geosci. 6, 701–710. Morel, F.M.M., Kustka, A.B., Shaked, Y., 2008. The role of unchelated Fe in the Fe nutrition of phytoplankton. Limnol. Oceanogr. 53, 400–404. Nagy, L., Yamaguchi, T., Yoshida, K., 2003. Application of EXAFS and XANES methods in coordination chemistry of carbohydrates and their derivatives. Struct. Chem. 14, 77–84. Nelson, D.M., Treguer, P., Brzezinski, M.A., Leynaert, A., Queguiner, B., 1995. Production and dissolution of biogenic silica in the ocean-revised global estimates, comparison with regional data and relationship to biogenic sedimentation. Glob. Biogeochem. Cycles 9 (3), 359–372. https://doi.org/10.1029/95GB01070. Quigg, A., 2016. Ch. 10 Micronutrients. In: Borowitzka, M.A., Beardall, J., Raven, J.A. (Eds.), The Physiology of Microalgae. vol 6. Springer, Dordrecht, pp. 211–231 Springer, Developments in Applied Phycology series. Quigg, A., Finkel, Z.V., Irwin, A.J., Rosenthal, Y., Ho, T.-Y., Reinfelder, J.R., Schofield, O., Morel, F.M.M., Falkowski, P.G., 2003. The evolutionary inheritance of elemental stoichiometry in marine phytoplankton. Nature 425, 291–294. Quigg, A., Irwin, A.J., Finkel, Z.V., 2011. Evolutionary imprint of endosymbiosis of elemental stoichiometry: testing inheritance hypotheses. Proc. R Soc. Biol. Sci. 278,

526–534. Quigg, A., et al., 2016. The role of microbial exoploymers in determining the fate of oil and chemical dispersants in the ocean. Limnol. Oceanogr. Lett. 1, 3–26. https://doi. org/10.1002/lol2.10030. Quigley, M.S., Santschi, P.H., Guo, L., Honeyman, B.D., 2001. Sorption irreversibility and coagulation behavior of 234Th with surface-active marine organic matter. Mar. Chem. 76, 27–45. Rijkenberg, M.J.A., Gerringa, L.J.A., Timmermans, K.R., Fischer, A.C., Kroon, K.J., Buma, A.G.J., Wolterbeek, B.T., de Baar, H.J.W., 2008. Enhancement of the reactive iron pool by marine diatoms. Mar. Chem. 109, 29–44. Rue, E.L., Bruland, K.W., 1997. The role of organic complexation on ambient iron chemistry in the equatorial Pacific Ocean and the response of a mesoscale iron addition experiment. Limnol. Oceanogr. 42 (5), 901–910. Schwehr, K.A., et al., 2018. Protein: polysaccharide ratio in exopolymeric substances controlling the surface tension of seawater in the presence or absence of surrogate Macondo oil with and without Corexit. Mar. Chem. 206, 84–92. Shaked, Y., Lis, H., 2012. Disassembling iron availability to phytoplankton. Front. Microbiol. 3, 123. https://doi.org/10.3389/fmich.2012.00123. Sreeram, K.J., Yamini Shrivastava, H., Nair, B.U., 2004. Studies on the nature of interaction of iron(III) with alginates. Biochim. Biophys. Acta 1670, 121–125. Sun, L., Chin, W.-C., Chiu, M.-H., Xu, C., Lin, P., Schwehr, K.A., Quigg, A., Santschi, P.H., 2019. Sunlight induced aggregation of dissolved organic matter: role of proteins in linking organic carbon and nitrogen cycling in seawater. Sci. Total Environ. 654, 872–877. Sutak, R., Botebol, H., Blaiseau, P.-L., Léger, T., Bouget, F.-Y., Camadro, J.-M., Lesuisse, E., 2012. A comparison study of iron uptake mechanisms in marine microalgae: iron binding at the cell surface is a critical step. Plant Physiol. 160, 2271–2284. Wu, J., Luther III, G.W., 1994. Size-fractionated iron concentrations in the water column of the western North Atlantic Ocean. Limnol. Oceanogr. 39 (5), 1119–1129. Xu, C., Zhang, S., Chuang, C.-Y., Miller, E.J., Schwehr, K.A., Santschi, P.H., 2011. Chemical composition and relative hydrophobicity of microbial exopolymeric substances (EPS) isolated by anion exchange chromatography and their actinide-binding affinities. Mar. Chem. 126 (1–4), 27–36. Xu, C., Zhang, S., Kaplan, D.I., Ho, Y.-F., Schwehr, K.A., Roberts, K.A., Chen, H., DiDonato, N., Athon, M., Hatcher, P.G., Santschi, P.H., 2015. Evidence for hydroxamate siderophores and other N-containing organic compounds controlling 239,240 Pu immobilization and re-mobilization in a wetland sediment. Environ. Sci. Technol. 49 (19), 11458–11467. Xu, C., Zhang, S., Sugiyama, Y., Ohte, N., Ho, Y.-F., Fujitake, N., Kaplan, D.I., Yeager, C., Schwehr, K.A., Santschi, P.H., 2016. Role of natural organic matter on iodine and 239,240 Pu distribution and mobility in environmental samples from the northwestern Fukushima Prefecture, Japan. J. Environ. Radioact. 2016 (153), 156–166. Xu, C., et al., 2018. The role of microbially-mediated exopolymeric substances (EPS) in regulating Macondo oil transport in a mesocosm experiment. Mar. Chem. 206 (20), 52–61. https://doi.org/10.1016/j.marchem.2018.09.005. Zhang, S., Xu, C., Santschi, P.H., 2008. Chemical composition and 234Th (IV) binding of extracellular polymeric substances (EPS) produced by the marine diatom Amphora sp. Mar. Chem. 112, 81–92. Zheng, J., Tagami, K., Uchida, S., 2013. Release of plutonium isotopes into the environment from the Fukushima Daiichi nuclear power plant accident: what is know and what needs to be know. Environ. Sci. Technol. 47, 9584–9595.

8