Characterization of IHSS Pony Lake fulvic acid dissolved organic matter by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and fluorescence spectroscopy Authors: Juliana D’Andrilli, Christine M. Foreman, Alan G. Marshall, & Diane M. McKnight. NOTICE: this is the author’s version of a work that was accepted for publication in Organic Geochemistry. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Organic Geochemistry, 65, December 2013. DOI# 10.1016/j.orggeochem.2013.09.013. D’Andrilli J, Foreman CM, Marshall AG, McKnight DM, "Characterization of IHSS Pony Lake fulvic acid dissolved organic matter by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and fluorescence spectroscopy," Org. Geochem 2013 65:19–28 Made available through Montana State University’s ScholarWorks scholarworks.montana.edu Characterization of IHSS Pony Lake fulvic acid dissolved organic matter by electrospray ion cyclotron resonance mass spectro spe Julia n McK a Cente SA b Ion C 0 E c Depar Way d INST A B S We pr e ( ic Substa ele ce mass s ix ce standard available through IHSS derived solely from a microbial source. A number of factors differentiate PLFA from other IHSS stan- dards, including source material, geographic tal influences. ESI FT-ICR MS and EEMS wer S Suwannee River (SR) FA, a standard frequent s = 0 or 1) constituents were present in both as dominated by CcHhOo compounds. Proteina ch we attributed to its microbial source materia th analytical techniques resulted in complementa ld be utilized for other microbiolog-ical environm Di is a s d from g biota d fatty y bioge d nutri e (Bern s bioge ing with respect to aq and for ide g the c The International Humic Substance Society (IHSS) was orga-nized in 1981 to promote education about instr l analysis of humic substances from specifically selected source materials (both solid and liquid phases // 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 biodeg-radation. 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 un-der all pH conditions and lighter in color, ranging from yellow to brown. IHSS standards are available for researchers to cr from various ana-lytical instruments and represent a DOM refer Many I N Titically examine humic substance experimental results ochemical reactivity as a function of chemical nature has yet to be resolved. Moreover, it is challeng uatic ecosystems to differentiate DOM derived from primarily autochthonous (microbial) sources allochthonous (soil and plant) material; investigating DOM from either source is advantageous hemical characteristics unique to various environments and ecosystems.ence for comparing and contrasting specific data sets with different DOM samples from other unique enviro researchers have focused on extensive DOM characterization by comparison with IHSSntifyin umenta ; http: the decay of OM composed of intact or remnant and transformed bio-molecules released from living and decayin (Mopper et al., 2007). It contains many identifiable classes of compounds such as sugars, amino acids, organic an acids, and humic sub-stances (Hansell and Carlson, 2001; Koch et al., 2005). Because it affects man ochemical processes in the environment (i.e. photochemical reactions, metal complexation, microbial growth, an ent and contaminant transport), determining its composi-tion is essential for understanding the global carbon cycl er, 1989). DOM derived from different sources (e.g. terrestrial, marine, glacial) has different properties, but itssolved organic matter (DOM) R O D U C T I O N location, sunlight exposure, freeze–thaw conditions, and other in situ environmen e used to compare the PLFA microbial DOM compositional signature with the IHS ly studied for environmental DOM analysis. Although CcHhOoNnSs (n = 0, 1, or 2 and IHSS samples, PLFA contained more N and S molecular species, whereas SRFA w ceous character was detected with both methods, in greater abundance for PLFA, whi l and labile, potentially more reactive nature than SRFA. Characterization from bo ry data that rein-force the importance of PLFA as an IHSS reference standard that shou ental DOM comparisons. ignificant component of marine and terrigenous aquatic ecosystems. It is formectroscopy na D’Andrilli a,⇑, Christine M. Forema night d r for Biofilm Engineering, Montana State University, Bozeman, MT 59717, U yclotron Resonance Program, National High Magnetic Field Laboratory, 180 tment of Chemistry & Biochemistry, Florida State University, 95 Chieftain AAR, University of Colorado, 1560 30th Street, Boulder, CO 80309, USA T R A C T esent the extensive characterization of Antarctic Pony Lak nce Society (IHSS) fulvic acid (FA) reference standard, using pectrometry (ESI FT-ICR MS) and excitation–emission matrization Fourier transform ion metry and fluorescence a, Alan G. Marshall b,c, Diane M. . Paul Dirac Drive, Tallahassee, FL 32310, USA , Tallahassee, FL 32306, USA PL) dissolved organic matter (DOM), an International Hum ctrospray ion-ization Fourier transform ion cyclotron resonan fluorescence spectroscopy (EEMS). PLFA is the first referennments. 2.4. Mass spectra and molecular formula assignmentstandards, specifically Suwannee River (SR, a terrestrial HA and FA analog). With the recent addition of Pony Lake (PL) FA, a microbi- ally 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 environ- ments 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 (77330S, 16690E) 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 perenni- ally 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 com- parisons with different environments. PLFA has been characterized by use of ion chromatography, organic carbon analysis, absorptiv- ity, molecular weight (MW) analysis, high performance size exclu- sion 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 hetero- geneous (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 exper- imenting with very limited/defined fractions not representative of the entire sample (Mopper et al., 2007). Bulk property measure- ments, while useful, cannot be used as true molecular descriptors because no ‘‘average’’ molecule within DOM defines its entire char- acter. No single analytical technique produces both bulk and de- tailed 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 resolu- tion and accuracy required to determine DOM molecular formulae over a wide range (200 750,000 at m/z 500) and accuracy (rms mass measure- ment error < 1 ppm). Instrumental parameters were selected for optimal natural OM MS acquisition and characterization. Excita- tion ranged from m/z 200–1500 at a frequency sweep rate of 50 Hz/ls and octopole frequency was maintained at 2.0 MHz. Mul- tiple (100–200) time domain acquisitions were co-added, Hanning apodized and zero-filled once before fast Fourier transformation and magnitude calculation (Marshall and Verdun, 1990).200 10,000 assignable peaks from one complex DOM spectrum. The signal/noise (S/N) threshold pro- vides a baseline that is sample specific, making it possible to char- acterize and compare natural OM more accurately. All molecular ions were determined to be singly charged by confirming the m/z 1.0034 spacing pattern between ions differing in elemental compo- sition by 12Cn and 12Cn1–13C1 (Limbach et al., 1991; Brown and Rice, 2000; Kujawinski et al., 2002). DOM characterization and molecular formula assignment from 9.4 T ESI FT-ICR MS analysis has been described in detail by Stenson et al. (2003). Briefly, large FT-ICR MS data sets (>10,000 peaks) are efficiently and reliably analyzed by conversion to Kendrick mass (Kendrick, 1963; Hughey et al., 2001; Stenson et al., 2003; D’Andrilli et al., 2010a). All possible elemental compositions within ± 1 ppm mass measurement error were con- sidered, subject to the following constraints appropriate for both PLFA and SRFA: 12C (0–100), 1H (0–200), 14N (0–2), 16O (0–30), 32S (0–2), and 13C (0–1). These compositional constraints were modeled after those reported by Stenson et al., (2003) for natural OM characterization and preliminary data from Brown et al., (2004), that described the elemental constituents C, H, N, O, and S that comprise PLFA samples and the structural functional group identification data regarding the N-containing species reported by Mao et al., (2007) and Fang et al., (2011). Specific restrictions were applied to eliminate potentially incor- rect formula assignments. Elemental composition was assigned to ion peaks for homologous series above the S/N threshold having specific mass error < 1 ppm, MS 13C peak confirmation and speci- fied mass spacing patterns. Negative ion peak masses were con- verted to neutral masses by addition of 1H+ = 1.007276 Da. The data were subsequently sorted by elemental composition (CcHhNn- OoSs), O content, degree of saturation and unsaturation, aromatic nature, or chemical heteroatom classification. Heteroatom content is defined as C, H, and O species (DOM molecular backbone) with varying numbers of N and/or S atoms. 2.5. Excitation–emission matrix fluorescence spectroscopy (EEMS) This approach has become relatively more common for probing the composition, concentration, and dynamics of fluorescent OM from various source materials (Coble, 1996; Hudson et al., 2007; Cory et al., 2010). EEMS scans report fluorescence intensity mea- sured over a range of excitation and emission wavelengths (Coble, 1996; Mopper et al., 1996; Hudson et al., 2007). Absorbance spectra (between 190 and 1100 nm) were collected for both samples by use of a Genesys 10 Series (Thermo-Scientific) Spectrophotometer (1 cm path length) to reduce inner filter effects during post processing (McKnight et al., 2001). Absorbance values were also monitored at 254 nm, a wavelength chosen to evaluate aromatic character of OM in natural waters (absorbance < 0.3 prior to EEMS; McKnight et al., 2001). PLFA values averaged A254 0.231 (n = 3), which is below the acceptable threshold, so no dilution was required. SRFA A254 values exceeded the threshold and thus were diluted accordingly to produce acceptable values that aver- aged A254 0.175 (n = 3). Dilution factors were recorded and in- cluded for post processing calculations. PLFA and SRFA EEMS spectra were obtained with a Horiba Jobin Yvon Fluoromax-4 Spectrofluorometer equipped with a Xe lamp light source and 1 cm path length quartz cuvette at 25 C with the following specifications: excitation wavelength 240–450 nm scanned over 10 nm intervals, emission wavelength 300–600 nm recorded in 2 nm increments, data integration period 0.25 s, 5 nm band pass for both excitation and emission monochromators; all data were generated in signal/reference mode to normalize the emission signal relative to the excitation light intensity.ized PLFA sample was comprised of only CcHhOo, the largest overall contribution to PLFA even though the proportion was considerably lower than the 84.5% value for SRFA. Both overall contributions of N and S containing species for PLFA molecular ions (58.8% and 38.1%) were strikingly different from SRFA (12.1% and 5.8%) and stressed the importance of a heteroatom contribution to the PLFAPost-collection data manipulation was performed in MATLAB to correct for inner filter effects, Raman scattering and a background blank Milli-Q Water subtraction. Position and intensity (Ex and Em maximum values) for individual fluorophores were determined to gain more information on the composition and character of PLFA and SRFA source material. 3. Results and discussion 3.1. 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 max- imize the production and detection of singly charged ions, reduce ion suppression effects, minimize ion collision and eliminate irre- producible ions between spectral scans (100–200 co-added MS data). Consequently, the data are more extensive in molecular composition coverage than any other single analytical technique. Moreover, our objective at the outset was to characterize the molecular composition of PLFA and subsequently to compare it with that of SRFA to provide further information on its constituents and advantage as an IHSS reference standard. Although the mass distributions for PLFA and SRFA spanned the same range (200 0.5) is based on Koch and Dittmar (2006).igned formulae in each category, carbon number ranges, and percent composition for RFA. PLFA and SRFA composition matches (%) Unique PLFA composition (%) Unique SRFA composition (%) 83.7 14.3 87.4 4.26 17.5 0 11.3 20.7 0 0.160 15.3 0 0.400 20.3 2.00 0.240 11.9 10.6 paced series in the m/z range ca. 0.25 window at H/C value ranging from 0.50–2.0. This region describes molecular ions significantly less oxygenated over varying levels of hydrogen saturation, and has been reported for other glacial and ice core DOM studies (Grannas et al., 2006; Bhatia et al., 2010; Singer et al., 2012; Stubbins et al., 2012). Major differences between PLFA and SRFA exist at this location and also at higher H/C ratio (1.5–2.0) over varying degrees of oxygenation, including molecular ions in each heteroatom class in the lipid-, protein-, amino sugar-, and cel- lulose-like regions. Variation in H/C and O/C data in the diagrams corresponds to different sources of OM between the two samples. PLFA is comprised of many chemical classes highlighted in Fig. 2, with noticeably more protein-, amino sugar-, cellulose-like, and more condensed aromatic species than SRFA. Protein-like DOM character has been shown to reflect more labile microbially influ- enced OM and Fig. 3a shows a significant contribution of protein- like species between H/C 1.5 and 2.0 and O/C 0.20 and 0.50, values not observed for SRFA. The occurrence of N-bearing species in the protein-like region was expected for microbially derived PLFA. We suggest that these compounds with lower MW (200 0.5) and the latter includes the possibility of CAO bonds as well as C@O bonds, yielding a low- er threshold for suggesting condensed aromatic structures (Koch and Dittmar, 2006). Note that this calculation is vital for visualizing unequivocal aromatic character from large DOM datasets based on low H/C nature, but involves some uncertainty regarding highly substituted aromatic species at AI values < 0.5 (examples discussed by Podgorski et al., 2012). Fig. 2 shows a solid black line that cor- responds to the conservative threshold of AI > 0.5 indicative of aro- matic structures and four downward pointing arrows that lead to more condensed aromatic species at lower H/C ratio (AIP 0.67; Koch and Dittmar, 2006). Overall, PLFA contains more highly con- densed aromatic compounds with varying amount of heteroatoms (CHO, CHON1, CHON2, CHON1S1 and CHON2S1) than SRFA (Fig. 3a). Fig. 3b shows SRFA condensed aromatic character for compounds containing only CHO and CHON1. Photochemical reactions may also explain some of the differ- ences between PLFA and SRFA. Gonsior et al. (2009) compared the nature of North Carolina River and estuary DOM before and after photo-irradation, displaying shifts to molecular ions with higher H/C ratio over a broad range of O/C ratio. The authors re- lated such shifts to the transformation of unsaturated/condensed aromatic compounds (based on AI values) to more saturated, less condensed aromatic molecules. We expected a higher abundance of condensed aromatic molecular data for SRFA than PLFA, for which less aromatic character has been reported (McKnight et al.,tained only CHO, CHOS1, CHON1S1, and CHON1 classes over H/C 1.20–1.27 and O/C 0.20–0.80 ranges (e.g., carbon number 15 and DBEs 7 in Fig. 4a and b). 3.4. EEMS: Source material, photooxidation and reactivity Complementary data sets from fluorescence spectroscopy are presented in Fig. 5a and b. Similar fluorescence patterns between PLFA and SRFA were observed in the humic-like fluorophore re- gions labeled A, M and C. We employed the labeled fluorophores A, C, B, T and M as proposed by Coble (1996). Table 3 includes a list of common natural water DOM fluorophores, descriptions and ob- served fluorescence regions for PLFA and SRFA. The unique fluores- cent nature of PLFA in Fig. 5a is evident at low excitation–emission wavelengths where proteinaceous fluorophores are present. Fluo- rescence in both B and T regions is characteristic of amino acid-like constituents in DOM derived from microbes, and are typically more labile in nature than humic substances. Both the proteina- ceous-like and more refractory humic-like fluorescence for PLFA agree with FT-ICR MS data as visualized from the van Krevelen dia- gram (Fig. 3a). Fig. 5a shows a shift to shorter wavelength (blue shift) for PLFA, which may be characteristic of less refractory hu- mic-like signatures compared with SRFA EEMS in Fig. 5b. According to the McKnight et al. (2001) fluorescence index (FI) calculation for uncorrected EEMS data, DOM having higher FI val- ues (1.8–1.9) corresponds to microbially derived OM, whereas ter- rigenous derived OM corresponds to lower FI values (1.3–1.4). For corrected EEMS data, the FI range was considerably broader for microbially derived than terrestrially derived OM (McKnight et al., 2001; Fulton et al., 2004; Schwede-Thomas et al., 2005). FI values calculated by the McKnight et al. (2001) method for PLFA and SRFA corrected EEMS were 1.45 ± 0.002 and 1.29 ± 0.011, with- in the range reported for these IHSS reference samples with differ- ent instruments (Cory et al., 2010). Note, the standard deviation was calculated for n = 3. Fig. 4. Isoabundance contoured plots of DBE vs. carbon number for (a) PLFA and (b) SRFA.Fig. 5. 3D EEMS and identified DOM fluorophores for IHSS samples (a) PLFA and (b)Biers et al. (2007) describe the role of N in developing chromo- phoric and fluorescent species in seawater DOM, but also summa- rize specific works that have confirmed in situ biological activity as a source of marine chromophoric DOM. Other reports confirm that N species are readily incorporated into humic substances in soil (Biers et al., 2007). Because dissolved organic N is susceptible to photochemical reactions (Bushaw et al., 1996; Reitner et al., 2002; Vähätalo and Zepp, 2005), we believe that N containing spe- cies are crucial for the differences between PLFA and SRFA compo- sition. Biers et al. (2007) found that microbial processing and photooxidation of dissolved organic and inorganic N compounds produced chromophoric DOM compounds. Therefore, we speculate that similar microbial processing of DOM is occurring for PLFA, producing some of the fluorescent characteristics observed with EEMS (Fig. 5a). N and S containing functional groups in FA DOM are especially important in determining the reactive nature of the DOM (Chin et al., 1994; McKnight et al., 2002). The amino acid-like fluoro- phores in the EEMS for PLFA suggest more reactive and labile OM than for SRFA, for which the contributions of the B and T peaks were far less evident. Therefore, we propose that the B and T re- gions correspond to some N and S containing species that also clus- ter in the more labile proteinaceous and amino sugar regions in the van Krevelen diagram for PLFA. N and S biogeochemistry occurring for PL and its link to phylogenetic data over the winter season in ice column samples was reported by Foreman et al., (2011). Sul- fur-reducing bacteria and sulfur-oxidizing chemolithoautotrophs were identified at PL, both of which involve oxidizing OM either by anaerobic respiration to produce H2S or by denitrification (reduction of oxidized N species by use of OM as electron donor) (Foreman et al., 2011). The authors reported a pungent odor of H2S at PL, supporting the occurrence of sulfate reduction by SRFA. Fluorescence intensity was normalized to the integral of the Raman peak from excitation at 350 nm and is given in Raman units (RU). 4. Conclusions et al ) 00 00 21 81High 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 constit- uents 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 pro- duced solely frommicrobial sources should soon become available. PLFA and SRFA samples contain components at varying levels of production and consumption, so the data here represent a mere snapshot of accumulated OM precursors, intermediates and prod- ucts. Comparing mass spectra and EEMS data gave the most infor- mation regarding the labile and recalcitrant nature of both DOM samples. Heteroatom content (N and/or S species) is a relatively new addition for analyzing the constituents of DOM with FT-ICR MS and has gained attention regarding degradation processes, pho-microbes and a potential shift from aerobic to anaerobic conditions with extensive OM uptake in the ice (Foreman et al., 2011). With identified MS compounds containing N and S and with existing data on microbial communities at PL (Foreman et al., 2011; Dieser et al., 2013), it is probable that the most labile material of PLFA originates from the degradation of longer chain proteins and from N and S chemical processes by microbial activity. We also specu- late that further transformation and reactions of microbially de- rived DOM biomolecules occur, producing refractory chemical species due to the data in and around the lignin-like region in the van Krevelen diagrams and humic-like fluorescence regions in the EEMS. However, the data regarding the exact transformation mechanisms of microbially derived DOM and their respective shifts to different DOM character (more humic-like) are considerably limited and further research is needed to understand the N and S microbiological influences on structural diversity, fluorescence and chemical nature of DOM. Table 3 Previously identified common natural water OM fluorophores (Coble, 1996; Marhaba SRFA. Fluorophore Description Ex/Em Max (nm A Humic-like 237–260/400–5 C Humic-like 300–370/400–5 M Marine humic-like 312/380–420 B Protein-like (tyrosine) 225–237/309–3 275/310 T Protein-like (tryptophan) 225–237/340–3 275/340tochemical affinity, source material and reactive character of the DOM. CcHhOoNnSs constituents are present in both PLFA and SRFA, but the proportion of each contributing class is different. PLFA is comprised of more labile, microbially derived material, reflected in the higher proportion of N and S containing species over a broad range of O/C and H/C ratios. This conclusion is supported by the presence of protein- and amino sugar-like components identified in the van Krevelen diagram and in the B and T fluorescent regions of the EEMS for PLFA, which were not observed for SRFA. We also report that both labile and refractory DOM constituents are present at PL, based on these data. At present, we can only speculate that the most labile constituents are proteinaceous compounds with varying heteroatom contributions derived from microbial metabo- lism intermediates or as end products. The more refractory mate- rial in PLFA (lignin-, cellulose- and humic-like signature regions) is indicative of degradation of microbially produced OM viademethylation and potentially dehydrogenation, as this environ- ment receives zero input from terrigenous sources. Both ESI FT-ICR MS and EEMS produced data that confirm the microbial source of PLFA. Further research is necessary, however, to determine the exact processes involved in microbial production and transformation of DOM, specifically regarding the pathways that may lead to lignin-like, more refractory DOM signatures in PL. However, the results stress the importance of incorporating PLFA as an IHSS standard that can be utilized for other glacial or microbially produced DOM comparisons with many environments. We currently use IHSS PLFA and SRFA as reference standards in other glacial DOM investigations to better understand microbe/ DOM interactions and terrestrial vs. microbially produced DOM signatures that can aid in identifying components in ice sheet and glacial ecosystems. 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 crea- tivity with the program MATLAB, P. Bloom and C. Zaccone at IHSS for website reference material assistance, and B. Vanmiddlesworth for the development of a PYTHON MS data analysis program. In addition, we thank two anonymous reviewers for their comments, suggestions and effort to greatly strengthen this work. The work was supported by the NSF Division of Antarctic Sciences through ANT-0338342 and ANT-0838970, by the NSF Division of Materials Research through DMR-11-57490, and the State of Florida. Appendix A. Supplementary material Supplementary data associated with this article can be found, ., 2000; Marhaba and Lippincott, 2000) and fluorescing regions assigned for PLFA and PLFA Ex/Em Maxima (nm) SRFA Ex/Em Maxima (nm) 240/440 240/450 320/425 320/450 310/400 310/425 240/300 240/300 270/310 – 240/340 240/340 270/340 –in the online version, at http://dx.doi.org/10.1016/j.orggeochem. 2013.09.013. Associate Editor—J.A. Rice References Berner, R.A., 1989. Biogeochemical cycles of carbon and sulfur and their effect on atmospheric oxygen over Phanerozoic time. Palaeography, Palaeoclimatology, Palaeoecology 73, 97–122. 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