Comparison of the Chemical Composition of Dissolved Organic Matter

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Comparison of the chemical composition of dissolved organic matter in three lakes in Minnesota, USA Xiaoyan Cao, George R. Aiken, Kenna Butler, Jingdong Mao, and Klaus Schmidt-Rohr Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04076 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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Environmental Science & Technology

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Comparison of the chemical composition of dissolved organic matter in three lakes in

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Minnesota, USA

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Xiaoyan Cao1,2, George R. Aiken3,+, Kenna D. Butler3, Jingdong Mao1,*, Klaus Schmidt-Rohr2

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Norfolk, Virginia 23529, USA

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02453, USA

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Department of Chemistry and Biochemistry, Old Dominion University, 4541 Hampton Blvd,

Department of Chemistry, Brandeis University, 415 South Street, Waltham, Massachusetts

United States Geological Survey, 3215 Marine Street, Boulder, Colorado 80303, USA

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Submitted to Special issue of Environmental Science & Technology honoring the

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contributions of George Aiken

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*Corresponding author:

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Jingdong Mao, e-mail: [email protected]; phone: 757-683-6874; fax: 757-683-4628

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Deceased.

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1 ACS Paragon Plus Environment


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Abstract

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New information on the chemical composition of dissolved organic matter (DOM) in three lakes

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in Minnesota has been gained from spectral editing and two-dimensional nuclear magnetic

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resonance (NMR) methods, indicating the effects of lake hydrological settings on DOM

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composition. Williams Lake (WL), Shingobee Lake (SL) and Manganika Lake (ML) have

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different source inputs, and the lake water residence time (WRT) of WL is markedly longer than

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that of SL and ML. The hydrophobic organic acid (HPOA) and transphilic organic acid (TPIA)

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fractions combined composed > 50% of total DOM in these lakes, and contained carboxyl-rich

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alicyclic molecules (CRAM), aromatics, carbohydrates, and N-containing compounds. The

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previously understudied TPIA fractions contained fewer aromatics, more O-rich CRAM, and

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more N-containing compounds compared to the corresponding HPOA. CRAM represented the

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predominant component in DOM from all lakes studied, and more so in WL than in SL and ML.

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Aromatics including lignin residues and phenols decreased in relative abundances from ML to SL

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and WL. Carbohydrates and N-containing compounds were minor components in both HPOA

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and TPIA and did not show large variations among the three lakes. The increased relative

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abundances of CRAM in DOM from ML, SL to WL suggested the selective preservation of

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CRAM with increased residence time.

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1. INTRODUCTION

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Substantial evidence has accumulated that lakes are hotspots of carbon cycling even though they

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comprise only a small fraction of the Earth’s surface.1-3 Dissolved organic matter (DOM) is the

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largest pool of organic carbon (OC) in most lake water, and contains OC both exported from

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land (allochthonous) and fixed by indigenous primary production (autochthonous). With the

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exception of eutrophic lakes, lake DOM is strongly dominated by allochthonous material

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imported from the catchment.4 A large share of lake DOM is altered and lost by in-lake

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processes including microbial respiration/mineralization, photochemical degradation, and

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flocculation.5-7 The magnitude of these in-lake processes appears to depend heavily on water

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residence time (WRT), i.e., the amount of time that water spends in the lake.8-12 For instance,

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Algesten et al.12 found that OC (mostly DOC) loss can be readily predicted from logWRT and

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increases rapidly with increasing WRT up to 2–3 years. Catalán et al.13 reported a negative

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relationship between the decay rate of OC in inland waters and WRT. Therefore, the molecular

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composition of lake DOM can be highly variable across landscapes, hydrologies, and climates,

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reflecting the net influences of DOM source, reactivity, and all the transformation processes

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occurring within the lake system.8, 9, 14-16

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The Shingobee River headwaters area, located in north-central Minnesota, provides a unique

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opportunity for comparing and contrasting two lakes (Williams Lake and Shingobee Lake) with

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similar biologic, geologic, and climatic settings, but different hydrologies.17 The hydrologically

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closed Williams Lake has no surface inlet or outlet. Groundwater inflow represents 58-76% of

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the annual water input with the rest being from precipitation.18 The hydrologically open

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Shingobee Lake, about 5 kilometers from Williams Lake, has the Shingobee River flowing



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through it and dominating the annual water flux to and from the lake. As a result, Shingobee

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Lake has a ten-fold shorter WRT (0.3-0.5 years) than Williams Lake (3-4 years).19

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A previous 13C nuclear magnetic resonance (NMR) study14 has shown that Williams Lake

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fulvic acid (FA; an operationally defined fraction of DOM isolated by XAD adsorption20) is

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more aliphatic and less aromatic than Shingobee Lake FA. The fluorescence characteristics of

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these FA isolates fell between the microbially-derived and terrestrially-derived end members,

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suggesting DOM sources from both microbially and terrestrially derived organic material.21

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Although the general chemistry of DOM in these two contrasting lakes has been reported in

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terms of DOC concentration, ultraviolet (UV) and fluorescence characteristics, and carbon

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functional group composition,14, 21 more detailed molecular-level structural information is

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lacking. Advanced NMR techniques, such as spectral editing and two-dimensional correlation,

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yield greater structural information beyond that which can be obtained from simply integrating

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the broad and overlapping NMR resonances. In addition, previous work has focused only on the

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relatively hydrophobic FA fraction of DOM (30-40% of total DOC),14, 21 whereas the more

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hydrophilic fraction (termed transphilic acid, TPIA), which represents an important piece of the

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DOC pie (~20% of total DOC), has received little attention. To extend this comparison, DOM

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samples from Manganika Lake, a hypereutrophic lake in northeastern Minnesota with

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anthropogenic influences from two major water inputs (the Virginia wastewater treatment plant

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and United Taconite mine waters) were also included. The residence time of water in Manganika

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Lake is estimated to be somewhat shorter than that of water in Shingobee Lake.

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The present study aimed to (1) provide detailed chemical-composition characterization of

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both hydrophobic acid and transphilic acid fractions of DOM from lakes with distinct

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hydrological conditions, using one- and two-dimensional solid-state NMR spectroscopy; and to 4 ACS Paragon Plus Environment

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(2) examine a previous conclusion8 that terrestrially derived DOM is selectively lost as residence

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time increases.

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2. MATERIALS AND METHODS

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2.1. Water Sampling and DOM Isolation. Sampling was conducted in June of 2012 in

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Manganika Lake (ML) in northeastern Minnesota, and in September of 2013 in Williams Lake

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(WL) and Shingobee Lake (SL) in north-central Minnesota. More detailed site descriptions can be

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found elsewhere.22, 23 Large volume (155-415 L) water samples were filtered (0.45 µm) in the

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field and shipped on ice to the U.S. Geological Survey (USGS) laboratory in Boulder (Colorado,

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United States) for DOC and UV absorbance analyses.

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Water samples were then processed by XAD isolation and concentration as described by

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Aiken et al.24 Briefly, samples were acidified to pH 2 with hydrochloric acid (HCl) and passed

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first through a column of XAD-8 resin, followed by a column of XAD-4 resin. Each column was

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then eluted with 0.1 M sodium hydroxide (NaOH) to obtain the XAD-8 (hydrophobic organic

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acids, HPOA) and XAD-4 (transphilic organic acids, TPIA) fractions. The eluates were

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immediately acidified to minimize sample alteration at high pH, desalted, lyophilized, and stored

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at room temperature.

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2.2. Elemental, UV-Visible Absorbance, and Carbon Isotopic Measurements. Elemental

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analyses (C, H, O, N, S and ash) of DOM isolates were performed by Huffman Laboratories

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(Golden, Colorado) using the method described by Huffman and Stuber.25 Specific UV

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absorbance (SUVA254) was determined by dividing the UV-visible absorbance at λ = 254 nm by

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DOC concentration, and is correlated to DOM aromaticity.26 Stable carbon isotope ratios (δ13C) 5 ACS Paragon Plus Environment


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were determined by isotope ratio mass spectrometry on dried DOM fractions following vapor

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phase acidification, and are expressed relative to the Pee Dee Belemnite (PDB) standard. The

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radiocarbon ratios of the HPOA isolates were measured by accelerator mass spectrometry at the

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Center for Accelerator Mass Spectrometry at Lawrence Livermore National Laboratory

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(California, United States). ∆14C data (in ‰) were corrected for isotopic fraction using measured

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δ13C values. The ∆14C and radiocarbon age were determined from percent modern carbon using

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the year of sample analysis according to Stuiver and Polach.27 Ages were presented as Modern

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when the fraction modern exceeded 1.

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2.3. NMR Analysis. All NMR experiments were performed at 100 MHz for 13C and 400

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MHz for 1H using a Bruker Avance 400 spectrometer equipped with a 4-mm double-resonance

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probe head. The 13C chemical shifts were referenced to tetramethylsilane, using the COO

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resonance of glycine in the α-modification at 176.49 ppm as a secondary reference. NMR

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experiments included quantitative multiple-cross polarization (multiCP),28 multiCP plus dipolar

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dephasing, cross-polarization with total suppression of sidebands (CP/TOSS), 13C chemical-

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shift-anisotropy (CSA) filter, 13C CSA filter plus dipolar dephasing, 1H–13C 2D heteronuclear

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correlation (HETCOR), 2D HECTOR with dipolar dephasing, and 2D HETCOR with 1H spin

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diffusion. Experimental details are described in the Supporting Information (SI) and a summary

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of these NMR methods and their purposes is provided in Table S1. The uncertainties of the NMR

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integrals (Table S2) were reported based on the propagation of analytical uncertainty, and

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estimated from the signal-to-noise ratio.

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3. RESULTS AND DISCUSSION 6 ACS Paragon Plus Environment

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3.1. Basic Chemical Properties. Table S3 lists DOC concentrations, SUVA254, elemental and

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carbon isotopic data of DOM from three lakes. DOC concentrations were greater in WL (7.2 mg

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C L-1) than in SL (5.2 mg C L-1), but lower than in ML (10.5 mg C L-1). The hydrophobic acids

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(HPOA) accounted for about 30-40% of DOC in all lakes, and were less abundant in WL (30% of

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DOC) than in SL (37%) and ML (40%). The fraction of transphilic acids (TPIA) was higher in

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WL (21% of DOC) and SL (22%) than in ML (17% of DOC). These observations are consistent

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with McElmurry et al.,29 who suggested that hydrologic “short-circuiting” may influence the

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hydrophobic/hydrophilic fractions of DOM. The SUVA254 (indicating aromaticity) associated

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with HPOA and TPIA isolates showed the same trend, increasing in values from WL to SL to ML;

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data generated during this study are available at https://doi.org/10.5066/F77M06VP.30 This result

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is consistent with the previously reported inverse relationship between DOC color (measured as

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absorbance) and WRT.31 The TPIA isolate always had a lower SUVA254 value than the

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corresponding HPOA from the same lake. Elemental analyses showed that N contents were low

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(< 2%) in all HPOA isolates, resulting in atomic C/N values of 36-37, but relatively higher (2-

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3%) in the TPIA fractions leading to atomic C/N values of 18-22. The δ13C values were less

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negative for WL HPOA (-26.1‰) than for SL HPOA (-29.1‰), but both fell within the range

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reported for DOC in lakes located in Sweden32 and Northern U.S.33, 34 The SL HPOA had more

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depleted ∆14C values than WL HPOA, which translated to 14C ages of 315 ybp for SL HPOA and

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modern for WL HPOA.

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3.2. Specific Functional Group Compositions of DOM in Different Lakes. A previous 13C

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NMR analysis of DOM in the Williams and Shingobee lake systems14 identified six types of

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functional groups, including aliphatic I carbon (0-62 ppm), aliphatic II carbon (62-90 ppm), 7 ACS Paragon Plus Environment


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acetal carbon (90-110 ppm), aromatic carbon (110-160 ppm), carboxyl carbon (160-190 ppm),

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and ketone C (190-230 ppm). The current study applied spectral-editing techniques to identify

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more specific functional groups and make more accurate assignments. For instance, multiCP

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spectra obtained after dipolar dephasing (Figure 1, red lines), showed signals of nonprotonated

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and mobile carbons, such as CH3, quaternary C (Cq), nonprotonated OC (OCnp), ketal C (OCnpO),

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nonprotonated aromatic C-C, aromatic C-O (144-160 ppm), COO/NC=O, and ketone C.

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Although both aldehyde and ketone C resonate in the 190-220 ppm region, only the signals of

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nonprotonated ketone C can survive dipolar dephasing. These dipolar-dephased spectra therefore

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provided supporting evidence for the previous assignment of the 190-230 ppm region to

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ketones;14 the nearly unchanged intensity of the ketone peak (in red lines) relative to that of the

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aldehyde/ketone peak (in black lines) indicated that the carbonyls were present as ketones, not

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aldehydes in all samples. In addition, there was a substantial peak at ~56 ppm in the dipolar-

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dephased spectrum of ML HPOA (Figure 1(c), red line), characteristic of methoxyl groups in

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lignin residues, but this sharp peak was missing in the spectra of all other samples. Moreover,

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signals of Cq and nonprotonated OC, indicative of highly branched structures, were prominent in

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the spectra of all lake isolates (Figure 1, red lines). Notably, the CSA-filtered spectra (Figure 1,

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bold black lines) resolved signals of di-oxygenated aliphatic carbons (O–C–O, see black arrows),

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which span the range of 100-123 ppm. The spectra obtained from the CSA filter with dipolar

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dephasing (Figure 1, blue lines) further indicated that nonprotonated di-oxygenated alkyl (OCnpO,

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see black arrows), i.e., ketal carbons, largely contributed to the NMR signals in the 100-123 ppm

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region. Therefore the previous assignment of 90-110 ppm only to acetal carbons (O–CH–O) was

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not appropriate.14


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Table 1 shows the distribution of the 13 specific functional groups in all HPOA/TPIA samples.

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The alkyl C (0-64 ppm) accounted for 34-47% and 33-41% of the C in the HPOA and TPIA

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isolates, respectively, and decreased in the order WL > SL > ML. The total aromatic C fraction

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(aromaticity; including protonated aromatic C-H, nonprotonated aromatic C-C, and oxygen

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substituted aromatic C-O) ranged from 12-27% and 8-16% for HPOA and TPIA isolates,

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respectively, and aromatic C-C dominated the aromatic C pool. The fractions of these three types

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of aromatic C generally displayed the same trend as aromaticity: WL < SL < ML. The abundances

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of O-alkyl C (sum of OC and OCO) remained rather constant among three lakes, accounting for

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19-20% of HPOA and 27-28% of TPIA. There were relatively more protonated OC (OCHn) than

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nonprotonated OC (OCnp), but fewer protonated OCO (OCHO, ~1%) than nonprotonated OCO

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(OCnpO, 2-3%). This suggested that these O-alkyl moieties were unlikely to be present in pure

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carbohydrate environments, where protonated OCH and OCHO are predominant. The

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COO/NC=O constituted 17-18% of HPOA and 20-21% of TPIA, with NC=O being at most 2-

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3% for HPOA and 4-5% for TPIA. Overall, HPOA isolates were more enriched in aromatics but

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depleted in O-alkyl carbons than corresponding TPIA isolates. This is consistent with the lower

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H/C and O/C atomic ratios of HPOA relative to the respective TPIA isolates, and with NMR

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measurements on HPOA/TPIA isolates from rivers and groundwater.24 The higher aromatic

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signature of HPOA isolates also agreed with previous findings that ~90% of the lignin phenols

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were recovered in the HPOA fractions.35, 36 Notably, all HPOA and TPIA samples contained

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abundant nonprotonated aliphatic C (Cq + OCnp + OCnpO) (11-13% for HPOA and 15-16% for

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TPIA) as well as CH3 (9-13%), indicating the presence of highly branched or cyclized aliphatic

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structures.

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3.3. Chemical Structures of Functional Groups in DOM from Different Lakes. Two-

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dimensional 1H-13C HETCOR NMR probes through-space 1H-13C correlations, identifies the

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chemical structure corresponding to a given carbon peak, and thus provides more information

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than 13C or 1H NMR alone.37 Moreover, the correlation of nonprotonated carbons with nearby

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(non-bonded) protons becomes possible, which enables characterization of the environment of

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nonprotonated groups. Particular emphasis is given to the chemical environment of O-alkyl C,

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COO, and abundant nonprotonated aliphatic carbons.

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Figure 2 presents the 2D HETCOR spectra of HPOA isolates from WL, SL and ML. For WL

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and SL, the 1H spectra associated with OC carbons contained signals from both O-alkyl protons

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and alkyl protons of similar intensities (Figure 2(d, e)). The OCO carbons showed cross peaks

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primarily with alkyl protons, indicating that both OC and OCO carbons were in close proximity

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to alkyl protons, and therefore, alkyl carbons. For ML HPOA (Figure 2(f)), both OC and OCO

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carbons correlated mainly with O-alkyl protons, but also more weakly with alkyl protons. The 1H

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spectra extracted at aromatic C (131 ppm) showed signals from both aromatic and alkyl protons

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(Figure 2(d-f)), indicating close association of aromatic and alkyl components. But the relative

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intensities of aromatic protons increased relative to those of alkyl protons from WL HPOA to SL

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HPOA and WL HPOA (Figure 2(d-f)). For all HPOA isolates, quaternary carbons (Cq) showed

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cross peaks mainly to alkyl protons (~2 ppm) (Figure 3(a-c)). The 1H spectra associated with

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OCnp (86 ppm) showed signals mainly from alkyl protons, but contributions from O-alkyl

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protons were also observed, and more pronounced for ML HPOA (Figure 3(c)) than WL and SL

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HPOA (Figure 3(a, b)). The ketal OCnpO (108 ppm) carbons appeared to associate primarily with

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alkyl protons for WL and SL HPOA. For ML HPOA, there were also contributions from O-alkyl

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protons and aromatic protons. These results suggested that nonprotonated OC and ketal carbons 10 ACS Paragon Plus Environment

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did not occur in carbohydrates where they would be surrounded by O-alkyl protons. The

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COO/NC=O carbons showed cross peaks primarily with alkyl protons near 2-3 ppm, with

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additional contribution from O-alkyl protons, indicating that they were attached mainly to alkyl

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and O-alkyl carbons, consistent with structures of carboxyl-rich alicyclic molecules (CRAM).

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The prominent cross peaks of COO carbons with the acidic COOH protons (~12 ppm) (Figure

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S1(a-c)) confirmed the presence of carboxylic acids rather than esters. Ketone C mainly

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correlated with aliphatic protons, indicating that they were bonded to aliphatic carbons. The 2D

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spectrum of ML HPOA also showed two distinct cross peaks of OCH3 (Figure S1(c)), red boxes):

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one of OCH3 carbon with its directly bonded protons, and the other of OCH3 carbon with

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aromatic protons. They confirmed the presence of lignin residues in ML HPOA.

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Unlike those of HPOA isolates, the 2D HETCOR spectra of TPIA isolates (Figure S2(a-c))

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showed pronounced cross peaks of OCO (~100 ppm) and OC (~72 ppm) with their directly

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attached protons, indicating the presence of sugar rings. The correlation of OC/OCO carbons

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with alkyl protons were much weaker for the TPIA (Figure S2(d-f)) than for the corresponding

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HPOA. With a 1-ms 1H spin diffusion time, the 1H spectra associated with OC carbons showed

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increasing signals from alkyl protons while those associated with alkyl C showed increasing

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contributions from O-alkyl protons (Figure S3(d-f)), compared to 1H spectra obtained without

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mixing time (Figure S2(d-f)). These trends indicated that alkyl and O-alkyl components were in

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close proximity and that sugar-ring structures did not form large carbohydrate domains in TPIA.

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There seemed to be two overlapping cross peaks of quaternary C near 45-60 ppm: one of Cq

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carbons with alkyl protons as similarly observed in spectra of HPOA samples (Figure S1), and

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the other of Cq carbons with OCH/NCH protons (Figure 4). The 1H slices associated with OCnp

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(~86 ppm) all showed major contributions from alkyl protons, but proximity to O-alkyl protons 11 ACS Paragon Plus Environment


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was also observed. The OCnpO carbons appeared to correlate primarily with alkyl protons in

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TPIA isolates from WL and SL (Figure S4(a,b)). For ML TPIA, the contribution from O-alkyl

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protons was equally, if not more important (Figure S4(c)). The 1H slices extracted at

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COO/NC=O (Figure S4) all showed a major band centered at 2-3 ppm and correlations with

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O/N-alkyl protons, suggesting that COO/NC=O groups are mostly attached to aliphatic carbons.

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The 1H slices extracted at ketone C for all TPIA isolates demonstrated that these carbons were

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attached to alkyl protons.

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3.4. New Structural Information on DOM in Lakes from Spectral Editing and 2D NMR.

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The main conclusion from previous NMR analysis of the Shingobee watershed is that “FA in WL

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is more aliphatic (0-62 ppm) and less aromatic (110-160 ppm) than FA samples from Shingobee

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Lake”.14 With spectral editing and two-dimensional NMR, the present study obtained new

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information and more structural details such as the makeup of aliphatic moieties. The structural

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components present in two major DOM fractions (HPOA and TPIA), which composed > 50% of

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total DOM in WL, SL and ML, included CRAM, aromatics, carbohydrates, and N-containing

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compounds. Their relative carbon percentages in HPOA/TPIA isolates were estimated following

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Cao et al.38 and are shown in Figure 5.

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Carbohydrates were a very minor component, contributing to ~ 5% in HPOA and 8% or less

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in TPIA isolates, respectively. This may seem surprising given that O-alkyl carbons (OC and

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OCO) accounted for ~20% of HPOA and ~28% of TPIA isolates, which would be traditionally

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attributed to carbohydrates. However, spectral editing and 2D NMR data showed strong evidence

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that approximately 1/3 to 1/2 of O-alkyl sites occurred as nonprotonated carbons close to alkyl

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protons (Figures 1, 3 and 4), indicating that they were unlikely present in carbohydrate 12 ACS Paragon Plus Environment

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environments. Even some protonated O-alkyl C (OCHn + OCHO) may not be completely

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associated with carbohydrates because they were also in close proximity to alkyl protons (Table

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1, Figures S3 and S5), which would not be expected in typical sugar environments. These results

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therefore caution against the routine assignment of 13C NMR resonances in 62-110 ppm to

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carbohydrates for DOM samples,39 in particular those isolated with solid-phase (e.g. XAD)

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extraction, which are known not to retain large carbohydrates.38

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CRAM represented the predominant structural component in both HPOA (61-76%) and TPIA

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(67-74%) isolates, but CRAM structures in TPIA are more oxygen-rich than CRAM in HPOA. A

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striking finding from our 2D NMR data is that abundant nonprotonated aliphatics (OCnp, OCnpO,

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and Cq, 11–13% of HPOA and 15–16% of TPIA) and isolated O–CH carbons were associated

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with CRAM structures in DOM from the three lakes. This characteristic of CRAM is also

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documented for DOM collected from the Yukon River, but has not been realized in other studies,

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where the analytical techniques employed were not capable of identifying these structural

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moieties.40-43

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Aromatics increased in abundance for both HPOA and TPIA from WL to SL and to ML

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(Figure 5), and their carbon percentages were linearly correlated to SUVA254 (r2 = 0.95, Figure

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S6). Characteristic peaks associated with lignin were evident only in ML HPOA. Furthermore,

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aromatic C abundances were lower in TPIA than in HPOA isolates. Based on C/N atomic ratios

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and the assumption that each N atom was attached to two C atoms, N-containing compounds (i.e.,

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carbons bonded to nitrogen) can account for up to 6% of HPOA isolates and 11% of TPIA

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isolates. There was little variation among the three lakes, but TPIA isolates contained more N

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than corresponding HPOA isolates.



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The more hydrophilic fraction, TPIA, which represents an important slice of the DOM pie

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(~20% of total DOC), has received little attention. Major differences between HPOA and TPIA

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fractions are that TPIA contains fewer aromatics, but more O-containing functional groups such

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as COO and O-alkyl moieties.24 More specifically from this study, these O-containing functional

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groups were mainly associated with CRAM structures, making CRAM in TPIA more oxygen-

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rich than CRAM in HPOA. Another difference was that N-containing compounds were more

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abundant in TPIA than in HPOA samples. The relatively higher aromatic content in HPOA than

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in TPIA fractions has been related to the 2−3 fold higher mercury methylation of the HPOA

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fraction compared to the TPIA fraction.44 On the other hand, molecules in the TPIA fractions,

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with their greater heteroatom and carboxyl contents, may exhibit considerable geochemical

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significance in processes such as mineral weathering and water acidification.

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3.5. Effects of Hydrology on DOM Structure in Lakes. Recent studies have explored the

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chemical composition of DOM from hundreds of Swedish lakes across a wide range of land-use,

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hydrology, and climate gradients, using fluorescence spectroscopy9 and mass spectrometry.8, 45

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The advanced NMR methods used in the present study can capture aspects of DOM composition

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that are missed by UV-Vis, fluorescence, or mass spectrometry, and they offer an important

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addition to current knowledge on DOM chemistry in lakes driven by hydrology. This work

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studied DOM from lakes in a small headwaters watershed in Minnesota on more localized scales,

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and allowed us to decipher the importance of lake hydrology in driving DOM chemistry.

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Lake hydrological settings influence the DOM source inputs, terrestrial/watershed processes

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that occur during DOM transport, and its in-lake processing,14, 29 which leads to the observed

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differences in DOM structure among the three lakes. DOM aromaticity decreased from 14 ACS Paragon Plus Environment

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Manganika Lake to Shingobee Lake and to Williams Lake. DOM from Manganika Lake was

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most enriched in aromatics, which were at least partially derived from lignin residues based on

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1D and 2D NMR data (Figures 1 and S1), while characteristic signals of lignin were not detected

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in Williams and Shingobee lakes. This agrees with the previous claim8 that terrestrially-derived

316

polyphenols were selectively lost as WRT increases. Yet WRT may not be the decisive factor in

317

determining the relative contribution of terrestrially-derived DOM. Although longer WRT may

318

facilitate the accumulation of autochthonous DOM9, 10 and in-lake photochemical processing of

319

terrestrially-derived aromatics,14 the influence of source materials cannot be neglected.14

320

Shingobee Lake receives terrestrial inputs from the Shingobee River, which supply more

321

aromatic DOM than groundwater entering Williams Lake.14 Manganika Lake receives yet more

322

terrestrial organic matter, from the Virginia wastewater treatment plant and United Taconite

323

mine waters. Therefore the lowest abundance of aromatics in Williams Lake among the three

324

lakes may also be attributed to the source input; WRT may be a correlate, and not fundamentally

325

associated with the specific process (e.g. photochemical transformation) responsible for the

326

variations in DOM observed. Furthermore, the influence of terrestrial processes that occur during

327

DOM transport can be also important. For instance, the absence of lignin observed in WL and SL

328

may also result from the removal of hydrophobic fractions during terrestrial/watershed processes

329

(rather than in-lake/autochthonous processes). Meier et al.46 reported the removal of more

330

aromatic and more hydrophobic components in DOM by sorption to soil mineral phases. Yano et

331

al.47 suggested DOM removal in soil occurs mostly via abiotic sorption. Kawahigashi et al.48

332

found that sorptive interactions of DOM with the soil mineral phase generally increase with

333

depth, and thus the depth of the active layer likely controls the quantity and quality of DOM

334

exported to aquatic systems. 15 ACS Paragon Plus Environment


335

In addition to supporting the previous conclusion that terrestrially-derived lignin is selectively

336

lost with increasing residence time,8 our study has also revealed DOM structures that may be

337

selectively preserved as residence time increases. Aliphatic components are most enriched in

338

DOM from Williams Lake, which could be attributed to the in-lake production of aliphatic

339

compounds,10 or an indirect consequence of the depletion of aromatic components. NMR data

340

clearly identified these aliphatic moieties to be mainly associated with CRAM. Note that neither

341

fluorescence nor mass spectrometry can differentiate CRAM from interfering molecules, such as

342

lignin, with similar elemental ratios.45 In addition to its photo-resistance, CRAM also contains

343

quaternary and nonprotonated O-alkyl carbons, which represent more humified components than

344

carbohydrates.49, 50 CRAM were a predominant component in DOM from all three lakes, and was

345

relatively more abundant in WL than in SL and ML (Figure 5). Therefore our results suggest the

346

selective preservation of CRAM with increasing water residence time.

347 348

3.6. Environmental Implications. Variations in the chemical composition of DOM in

349

aquatic systems, as observed from this study, are significant factors controlling DOM chemical

350

reactivity. For instance, the aromatic carbon content in DOM influences the strength of DOM

351

interactions with organic pollutants. Graham et al.44 reported that highly aromatic DOM strongly

352

enhanced mercury methylation, consistent with previous identification of environments with

353

high concentrations or fluxes of highly aromatic DOM (e.g., wetlands) as hot spots for

354

methylmercury production.51 Aromatic moieties are photoreactive and play mechanistic roles in

355

DOM photoreactions, including oxidation/reduction reactions that control the speciation and

356

chemistry of metals such as Fe and Hg.52 The globally ubiquitous, quantitatively important

357

component of DOM, CRAM, has been predicted to be microbially and photochemically 16 ACS Paragon Plus Environment

Page 16 of 28

Page 17 of 28


358

refractory. This stable nature is further supported by our observation that CRAM are selectively

359

preserved with increased lake WRT, suggesting their critical role in the global carbon cycle.

360

Nevertheless, whether CRAM are closely tied to biogeochemical processes such as interactions

361

with inorganic and organic pollutants remains unknown and warrants further investigations.

362 363 364

Acknowledgments This work was funded by USDA-NIFA Capacity Building Grant Program (grant 2010-38821-

365

21558), National Aeronautics and Space Administration (grants NNX09AU89G and

366

NNH04AA62I), the National Science Foundation (grants CBET-0853950 and CBET-0853682)

367

and the U.S Geological Survey National Research Program. Any use of trade, firm, or product

368

names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

369 370

Supporting Information

371

The Supporting Information is available free of charge on the ACS Publications website.

372

More NMR experimental details. Tables summarizing the NMR methods and their purposes, as

373

well as the uncertainties of the NMR integrals. A table containing DOC concentrations, SUVA254,

374

elemental and carbon isotopic data. Figures showing the 2D 1H-13C HETCOR NMR spectra and

375

the 1H slices extracted from the 2D spectra. The relationship between percent aromaticity

376

determined by 13C NMR and SUVA254.

377



378

References

379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

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43. Zigah, P. K.; Minor, E. C.; Abdulla, H. A.; Werne, J. P.; Hatcher, P. G., An investigation of size-fractionated organic matter from Lake Superior and a tributary stream using radiocarbon, stable isotopes and NMR. Geochim. Cosmochim. Acta 2014, 127, 264-284. 44. Graham, A. M.; Aiken, G. R.; Gilmour, C. C., Effect of dissolved organic matter source and character on microbial Hg methylation in Hg–S–DOM solutions. Environmental Science & Technology 2013, 47, (11), 5746-5754. 45. Kellerman, A. M.; Kothawala, D. N.; Dittmar, T.; Tranvik, L. J., Persistence of dissolved organic matter in lakes related to its molecular characteristics. Nature Geoscience 2015, 8, (6), 454-457. 46. Meier, M.; Chin, Y.-P.; Maurice, P., Variations in the composition and adsorption behavior of dissolved organic matter at a small, forested watershed. Biogeochemistry 2004, 67, (1), 39-56. 47. Yano, Y.; Lajtha, K.; Sollins, P.; Caldwell, B. A., Chemistry and dynamics of dissolved organic matter in a temperate coniferous forest on andic soils: effects of litter quality. Ecosystems 2005, 8, (3), 286-300. 48. Kawahigashi, M.; Kaiser, K.; Kalbitz, K.; Rodionov, A.; Guggenberger, G., Dissolved organic matter in small streams along a gradient from discontinuous to continuous permafrost. Global Change Biology 2004, 10, (9), 1576-1586. 49. Mao, J. D.; Kong, X. Q.; Schmidt-Rohr, K.; Pignatello, J. J.; Perdue, E. M., Advanced solid-state NMR characterization of marine dissolved organic matter isolated using the coupled reverse osmosis/electrodialysis method. Environmental Science & Technology 2012, 46, (11), 5806-5814. 50. Helms, J. R.; Mao, J.; Stubbins, A.; Schmidt-Rohr, K.; Spencer, R. G.; Hernes, P. J.; Mopper, K., Loss of optical and molecular indicators of terrigenous dissolved organic matter during long-term photobleaching. Aquatic Sciences 2014, 353-373. 51. Aiken, G. R.; Gilmour, C. C.; Krabbenhoft, D. P.; Orem, W., Dissolved organic matter in the Florida Everglades: implications for ecosystem restoration. Critical Reviews in Environmental Science and Technology 2011, 41, (S1), 217-248. 52. Aiken, G., 1.11 Dissolved Organic Matter in Aquatic Systems. In Comprehensive Water Quality and Purification, Ahuja, S., Ed. Elsevier: Waltham, Massachusetts, 2014; Vol. 1, pp 205-220.

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TPIA

HPOA

Table 1. Peak areas (in %) in 13C multiCP NMR spectra of HPOA and TPIA isolates from different lakes, and the assigned structural moieties associated with the spectral regions. ppm 190160220-190 143-100 123-100 (OCO) 100-64 (OC) 64-0 (Alkyl C) Samples 160 143 Ketone COO/ Arom. Arom. Arom. CH2/CH/ OCnpOb OCHOb OCnpa OCHn Cqa OCH3a CH3a a C NC=O C-O NCH C-C C-H

a b

Williams

1.9

17.4

3.0

5.6

4.1

1.8

0.9

6.4

11.0

5.2

0.1

29.0

13.5

Shingobee

2.6

18.3

4.5

10.0

5.0

2.1

0.8

7.0

9.8

4.4

0.9

23.1

11.5

Manganika

2.5

16.9

6.7

13.2

7.2

2.1

0.8

6.1

10.3

3.1

1.6

20.4

9.3

Williams

1.3

20.8

2.4

3.6

2.4

2.4

1.1

8.9

15.9

4.0

< 0.1

26.8

10.4

Shingobee

2.4

21.3

3.2

5.9

3.2

2.9

0.9

9.3

14.9

3.8

< 0.1

23.3

8.9

Manganika

2.6

20.0

4.2

8.6

3.2

2.8

1.4

9.4

14.2

3.6

< 0.1

21.2

8.8

Based on a multiCP spectrum with 40-µs dipolar dephasing. Based on a CSA-filtered CP/TOSS spectrum and a CSA-filtered CP/TOSS spectrum with dipolar dephasing.

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Figure Captions Figure 1. Solid-state 13C multiCP NMR spectra and spectral editing for identification of specific functional groups of HPOA and TPIA isolates from Williams Lake (a and d), Shingobee Lake (b and e) and Manganika Lake (c and f). Thin black lines: MultiCP spectra showing signals of all C. Red lines: MultiCP with dipolar dephasing showing nonprotonated C and mobile segments such as CH3. Bold black lines: selection of sp3-hybridized C signals by a 13C CSA filter for the separation (see arrows) of O-C-O from aromatic C. Blue lines: selection of nonprotonated sp3hybridized C signals and mobile segments by CSA filter with dipolar dephasing for the separation of nonprotonated O-C-O (shaded area) from nonprotonated aromatic C. The multiCP spectra were scaled to give the same intensity of the COO/NC=O band. Figure 2. 2D 1H-13C HETCOR spectra with 0.5-ms HH-CP of HPOA isolates from (a) Williams Lake, (b) Shingobee Lake and (c) Manganika Lake. 1H slices extracted from the 2D spectra: (d) refers to 1H slices of spectrum (a), (e) 1H slices of spectrum (b), and (f) 1H slices of spectrum (c). Figure 3. 1H slices extracted from the 2D 1H-13C HETCOR spectrum with 0.5-ms HH-CP and 40-µs dipolar dephasing of HPOA isolates from (a) Williams Lake, (b) Shingobee Lake, and (c) Manganika Lake. Figure 4. 2D 1H-13C HETCOR spectrum with 0.5-ms HH-CP and 40-µs dipolar dephasing of TPIA isolates from (a) Williams Lake, (b) Shingobee Lake, and (c) Manganika Lake. Figure 5. Relative carbon percentages of the four compound classes (carbohydrates, CRAM, aromatics, and N-containing) within HPOA and TPIA isolates in Williams Lake, Shingobee Lake, and Manganika Lake.



Figure 1.


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Page 25 of 28


Figure 2.



Figure 3.

Figure 4.


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Page 27 of 28


Figure 5.



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Comparison of the Chemical Composition of Dissolved Organic Matter

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