Detection of carbohydrates in sea ice extracellular polymeric substances via solid-phase extraction and HPLC-ESI-MS/MS
Introduction
Sea ice is a complex matrix containing channels, capillaries and pores, closely connected with the water column below, and it is characterized by steep gradients in temperature, salinity, light and nutrient concentrations. Nevertheless, different microbial communities, known as the sympagic biota, are able to survive in the brine inclusions and interstices of the sea ice habitat (Saggiomo et al., 2016). The most conspicuous members of the sea ice communities are the microalgae that have adapted to live in extreme conditions and flourish within the distinct micro-habitats that are created when the sea ice forms and develops (Saggiomo et al., 2016). These microalgae can influence the development of ice edge blooms in the water column by providing an inoculum or “seed population” (Grotti et al., 2005).
High concentrations of dissolved organic matter (DOM), dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) are usually associated with the biological assemblages in sea ice. Several studies have demonstrated that DOM is rich in extracellular polymeric substances (EPS) secreted in the form of mucous gels by sea ice algae and bacteria for cryoprotection, halotolerance, attachment to substrate, nutrient uptake and formation of chains, colonies and biofilm (Krembs and Deming, 2008; Juhl et al., 2011; Hassler et al., 2011a, Hassler et al., 2011b). EPS are complex macromolecules ranging from the dissolved phase through the colloidal and include lipids, nucleic acids, proteins and carbohydrates. In particular, carbohydrates are the most abundant component, generally representing 40 to 95% of the EPS in marine autotrophic biofilms (Mancuso et al., 2004; Underwood et al., 2010).
Recent evidence has indicated that EPS may contribute to the Fe-binding organic ligand pool (Lorg), increasing the residence time of bioavailable Fe in the euphotic zone, therefore possibly sustaining and controlling primary productivity in sensitive oceanic regions, such as the Southern Ocean (Hassler et al., 2011a). In fact, in polar areas, sea ice is fundamental in the biogeochemical cycle of Fe (Sedwick and Ditullio, 1997), as it accumulates and stores Fe during winter months and releases it to surface waters after its melting during spring/summer months (Lannuzel et al., 2015). Despite the ecological importance of EPS and of their associated carbohydrates in particular, there is limited information in the literature regarding their chemical characterization, due to their heterogeneous composition, complex structures and low concentrations in a matrix with a high ionic strength (Gledhill and Buck, 2012).The majority of studies characterizing the distribution of sea ice EPS were performed by colorimetric or spectrophotometric assays, following in several cases an EPS size fractionation (Underwood et al., 2010; Aslam et al., 2012). More information was obtained by Hassler et al. (2011a) using a flow field-flow technique fractionation coupled to multiple detectors.
The objective of this work is the development of a suitable analytical method for the identification of the chemical composition of dissolved EPS in sea ice to obtain information which cannot be inferred by spectrophotometric methods or other techniques such as the competitive ligand equilibration - adsorptive cathodic stripping voltammetry (CLE-AdCSV).
In particular, high performance liquid chromatography - electrospray ionization - tandem mass spectrometry (HPLC-ESI-MS/MS) was considered, since this technique allows the separation of the analytes from the interfering compounds, it is sensitive and it provides structural information following the fragmentation of the parent ion and the isotopic distribution, that can be used to identify specific elements in a molecule (Mccormack et al., 2003).
In this paper we propose a new method for the determination of EPS in sea ice samples, using Solid Phase Extraction (SPE) for the extraction and the pre-concentration steps and HPLC-ESI-MS/MS for the separation and identification of the analytes. In particular, we developed the extraction and pre-concentration procedure from smaller volumes of samples than those reported in previous methods (McCormack et al., 2003; Dittmar et al., 2008; Mawji et al., 2008; D'Andrilli et al., 2010; Velasquez et al., 2011). Moreover, we used the versatility and efficiency of HPLC-MS/MS in order to obtain more information about the molecule structures of the extracted compounds.
Section snippets
Sample collection and processing
The method has been optimized using composite samples obtained from pack-ice aliquots collected during winter 2012 in the framework of the activities of Australian-led Sea Ice Physics and Ecosystem eXperiment-2 (SIPEX-2) voyage.
Pack-ice samples were collected at six stations during the SIPEX-2 survey in Austral winter/spring 2012 (26 Sep–28 Oct, 64.26–65.15°S/118.55–120.58°E) (Fig. 1). At each station, between 5 and 8 sea ice cores were collected for a suite of physical and biogeochemical
Results
The chromatogram obtained by preconcentration of 500 mL of the sample showed a high total ionic current (Fig. 3). The sample was then diluted 1:10 and 1:20, before its introduction into the HPLC system to evaluate significant changes in the intensity of the signal. Despite the dilutions, the chromatograms maintained a high intensity of the signal, that allowed us to use a smaller sample volume in the SPE extraction step. The total ionic current (TIC) measured for the 50 mL sample was high
Discussion
EPS are an important component of the DOM in the sea ice, playing several biological roles (Krembs et al., 2002). Moreover, they have affinity for Fe and they can influence its biogeochemical cycle, speciation and bioavailability (Gledhill and Buck, 2012).
The sample preparation and subsequent analysis of EPS are difficult, due to their heterogeneous composition, complex structures and low concentrations in a matrix with a high ionic strength (Gledhill and Buck, 2012). Therefore, large sample
Conclusion
HPLC-ESI-MS/MS proves to be a powerful technique for the separation and identification of EPS in sea ice samples. Despite its sensitivity, a SPE preconcentration is necessary for the detection of these compounds occurring in samples at ppb levels. Nevertheless, major advantages over previously published approaches include a smaller volume of the sample and the ability to obtain structural information on EPS. Nine EPS having a mass lower than 1500 Da were identified by HPLC-ESI-MS/MS,
Declaration of Competing Interest
None.
Acknowledgements
The authors would like to thank Dr. Delphine Lannuzel (Institute for Marine and Antarctic Studies, Hobart, Tasmania, Australia) for providing us with the sea ice samples collected in the framework of SIPEX 2 project (Australian Research Council (LE0989539 and DE120100030), the Australian Government Cooperative Research Centers Programme through the Antarctic Climate & Ecosystems (ACE CRC, ACE-Carbon 2.1) and the Australian Antarctic Science (AAS) project n. 4051).We are grateful to Dr. Silvia
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