Characterization of IHSS Pony Lake fulvic acid dissolved organic matter by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and fluorescence spectroscopy
Introduction
Dissolved organic matter (DOM) is a significant component of marine and terrigenous aquatic ecosystems. It is formed from the decay of OM composed of intact or remnant and transformed biomolecules released from living and decaying biota (Mopper et al., 2007). It contains many identifiable classes of compounds such as sugars, amino acids, organic and fatty acids, and humic substances (Hansell and Carlson, 2001, Koch et al., 2005). Because it affects many biogeochemical processes in the environment (i.e. photochemical reactions, metal complexation, microbial growth, and nutrient and contaminant transport), determining its composition is essential for understanding the global carbon cycle (Berner, 1989). DOM derived from different sources (e.g. terrestrial, marine, glacial) has different properties, but its biogeochemical reactivity as a function of chemical nature has yet to be resolved. Moreover, it is challenging with respect to aquatic ecosystems to differentiate DOM derived from primarily autochthonous (microbial) sources and allochthonous (soil and plant) material; investigating DOM from either source is advantageous for identifying the chemical characteristics unique to various environments and ecosystems.
The International Humic Substance Society (IHSS) was organized in 1981 to promote education about instrumental analysis of humic substances from specifically selected source materials (both solid and liquid phases; http://www.humicsubstances.org/). Humic substances are the product of biogeochemical degradation of detrital biomass and are considered to be the most refractory component of DOM with respect to its resistance to further biodegradation. Aqueous humic substances can be subdivided into two fractions: (i) humic acid (HA) – the major extractable component of humic substances which are dark brown in color and not soluble in water below pH 2, and (ii) fulvic acid (FA) – soluble in water under all pH conditions and lighter in color, ranging from yellow to brown. IHSS standards are available for researchers to critically examine humic substance experimental results from various analytical instruments and represent a DOM reference for comparing and contrasting specific data sets with different DOM samples from other unique environments. Many researchers have focused on extensive DOM characterization by comparison with IHSS standards, specifically Suwannee River (SR, a terrestrial HA and FA analog). With the recent addition of Pony Lake (PL) FA, a microbially derived DOM reference sample potentially analogous to other microbially dominated environments is available.
The global reservoir of DOM is substantial: its amount is greater than the quantity of carbon stored as CO2 in the atmosphere (Gorham, 1991, Hedges, 2002). Marine and terrestrial aquatic systems are thought to be the largest contributors; however, a third reservoir exists that functions as both a source and sink for natural OM, i.e. ice. Until recently, it was believed that glacial environments were devoid of life, but many discoveries of microbial communities and OM in glacial systems have generated attention toward a better understanding of life in these extreme environments and, in turn, studying its contribution to the global carbon cycle (Sharp et al., 1999, Priscu and Christner, 2004, Priscu et al., 2008, Lanoil et al., 2009, Foreman et al., 2011; D’Andrilli et al., unpublished results).
The four main locations for IHSS HA and FA standards are SR (river water in Okefenokee Swamp, Georgia), Elliot Soil (fertile prairie soils, Illinois), Pahokee Peat (agricultural peat soil, Florida Everglades) and Leonardite (a low grade coal, North Dakota), all of which are found in the continental USA and are heavily exposed to higher plant/terrestrial input (site information available at http://www.humicsubstances.org/). PL (77°33′S, 166°9′E) is a coastal pond on Cape Royds, Ross Island, Antarctica. Completely isolated from terrigenous input and with no existing higher plants in the watershed, PL represents an excellent system for studying autochthonous, microbially derived DOM; hence its appeal as an IHSS standard FA sample. The lake is shallow (1–2 m) and perennially ice covered, but areas may melt during the austral summer. It contains a relatively high concentration of DOM (1.3–2.4 mM), compared with other glacial environments, and sustains living organisms such as bacteria, virus-like particles, algae and ciliate protozoans throughout the year (Brown et al., 2004, Foreman et al., 2011, Dieser et al., 2013). It provides a DOM FA reference standard that adds to the repertoire available for future DOM comparisons with different environments. PLFA has been characterized by use of ion chromatography, organic carbon analysis, absorptivity, molecular weight (MW) analysis, high performance size exclusion chromatography, 13C nuclear magnetic resonance spectroscopy (Brown et al., 2004) and excitation–emission matrix fluorescence spectroscopy (EEMS; Cory et al., 2010). However, no detailed molecular qualitative information has been provided for this IHSS standard.
The task of identifying DOM molecular components presents a significant challenge because they are polyfunctional and heterogeneous (containing C, H, N, O and S), and vary greatly in MW and concentration (Mopper et al., 2007). Analyzing DOM has until recently been limited to characterizing its bulk properties or experimenting with very limited/defined fractions not representative of the entire sample (Mopper et al., 2007). Bulk property measurements, while useful, cannot be used as true molecular descriptors because no “average” molecule within DOM defines its entire character. No single analytical technique produces both bulk and detailed molecular information regarding DOM, so it is common for multiple techniques to be applied.
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) at high magnetic field (> 7 T) (Marshall et al., 1998; Marshall and Rodgers, 2008) is unrivaled for achieving the resolution and accuracy required to determine DOM molecular formulae over a wide range (200 < m/z < 1000). It is possible to characterize DOM samples from different sources by determining the molecular constituents, as applied for extensive characterization and comparison of other environmental DOM samples with SRFA (Freitas et al., 2001, Kujawinski et al., 2002, Stenson et al., 2003, Sleighter and Hatcher, 2007; Bhatia et al., 2010). We report here complementary data (from FT-ICR MS and EEMS) for advanced and bulk DOM characterization from this unique Antarctic system in order to highlight PLFA as a useful IHSS reference standard by comparing it with SRFA.
Section snippets
Sampling
PLFA and SRFA reference standards were obtained from the IHSS. Chemical information and source material description can be found at http://www.humicsubstances.org/. The samples were selected for two reasons: (i) PLFA has not been characterized at the molecular level with FT-ICR MS and represents an excellent reference for microbially derived DOM from other glacial, marine and terrigenous ecosystems and (ii) SRFA represents an excellent comparative DOM sample as it has been extensively
ESI FT-ICR MS of PLFA and SRFA
ESI 9.4 T FT-ICR spectra of PLFA and SRFA are shown in Fig. 1a and b. Although we acknowledge the selectivity effects in the methodology for isolation of both PLFA and SRFA IHSS samples and ESI FT-ICR MS, instrumental parameters were chosen to maximize the production and detection of singly charged ions, reduce ion suppression effects, minimize ion collision and eliminate irreproducible ions between spectral scans (100–200 co-added MS data). Consequently, the data are more extensive in molecular
Conclusions
High magnetic field ESI FT-ICR MS and EEMS are important tools for complementary advanced and bulk characterization of natural OM. IHSS standard and reference samples have been used in the past to compare and contrast different types of DOM constituents with other natural samples in order to better understand its chemical characterization. Most commonly used is SRFA; however, with the addition of PLFA, more information regarding DOM produced solely from microbial sources should soon become
Acknowledgements
Mass spectra were obtained at the National High Magnetic Field Laboratory in Tallahassee, Florida and EEMS data were generated at the Center for Biofilm Engineering at Montana State University, Bozeman, Montana. We thank D. Podgorski and the Ion Cyclotron Resonance Facility staff for maintaining the optimal performance of the 9.4 T FT-ICR mass spectrometer. We acknowledge K. Hunt and T. Vose for their extensive knowledge, code-writing and creativity with the program MATLAB, P. Bloom and C.
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