The Structural Arrangement and Relative Abundance of Aliphatic Units

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Environmental Measurements Methods

The Structural Arrangement and Relative Abundance of Aliphatic Units May Effect Long-Wave Absorbance of Natural Organic Matter as Revealed by 1H NMR Spectroscopy Irina V. Perminova, Evgeny A. Shirshin, Andrey I. Konstantinov, Alexander Ya. Zherebker, Ivan V. Dubinenkov, Vasily A. Lebedev, Natalia A. Kulikova, Eugene N. Nikolaev, Ekaterina Bulygina, and Robert Max Holmes Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01029 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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Environmental Science & Technology

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The Structural Arrangement and Relative Abundance of Aliphatic Units May

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Effect Long-Wave Absorbance of Natural Organic Matter as Revealed by 1H

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NMR Spectroscopy

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I. V. Perminova1*,

E.A. Shirshin,2

A. I. Konstantinov,1

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I. V. Dubinenkov,1 N. A. Kulikova, 1,6,7 E.N. Nikolaev,3,4 E. Bulygina,8 R. M. Holmes8

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1

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Moscow, Russia; 2Department of Physics, Lomonosov Moscow State University, Leninskie Gory 1-

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2, 119991 Moscow, Russia; 3Skolkovo Institute of Science and Technology, 143025 Skolkovo,

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Moscow region, Russia, 4Institute for Energy Problems of Chemical Physics of RAS, Leninskij pr.

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38 - 2, 119334 Moscow, Russia 5Department of Materials Science, Lomonosov Moscow State

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University, Leninskie Gory 1-73, 199991 Moscow, Russia;

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Lomonosov Moscow State University, Leninskie Gory 1-12, 199991 Moscow, Russia; 7Bach

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Institute of Biochemistry of RAS, Federal Research Center “Biotechnology”, Leninskij pr. 33–2,

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119071 Moscow, Russia 8Woods Hole Research Center, 149 Woods Hole Rd, Falmouth,

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Massachusetts 02540, United States

V. A. Lebedev,1,5

Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1-3, 119991

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A. Zherebker,1,3,4

*E-mail: [email protected]

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ACS Paragon Plus Environment

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Department of Soil Science,


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Abstract

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The objective of this study was to shed light on structural features which underlay intensity of long

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wave absorbance of natural organic matter (NOM) using 1H NMR spectroscopy. For this purpose, a

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set of the NOM samples was assembled from arctic and non-arctic sampling sites (the Kolyma river

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basin and Moscow region, respectively). It was to assure a substantial difference in the humification

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degree of the isolated organic matter - the biogeochemical proxy of the long-wave absorbance of

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NOM. The assembled NOM set was analyzed using solution-state 1H NMR spectroscopy. The

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distribution of both backbone and exchangeable protons was determined using acquisition of spectra

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in three different solvents. The substantially higher contribution of non-functionalized aliphatic

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moieties CHn (e.g., materials derived from linear terpenoids, MDLT) in the arctic NOM samples

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was revealed as compared to the non-arctic ones. The latter were characterized with the higher

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content of CHα protons adjacent to electron-withdrawing groups which belong to carboxyl rich

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alicyclic moieties (CRAMs) or to aromatic constituents of NOM. We have calculated a ratio of CHn

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to CHα protons as a structural descriptor which showed significant inverse correlation to intensity of

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long wave absorbance assessed with a use of E4/E6 ratio and the slope of absorption spectrum. The

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steric hindrance of aromatic chromophoric groups of the NOM ensemble by bulky non-

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functionalized aliphatic moieties (e.g., MDLT) was set as a hypothesis for explanation of this

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phenomenon. The bulky aliphatics might increase a distance between the interacting groups resulting

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in inhibition of electronic (e.g., charge-transfer) interactions in the NOM ensemble. The obtained

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relationships were further explored using Fourier transform mass spectrometry as complementary

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technique to 1H NMR spectroscopy. The data obtained on correlation of molecular composition of

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NOM with 1H NMR data and optical properties were very supportive of our hypothesis that

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capabilities of NOM ensemble of charge transfer interactions can be dependent on structural

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arrangement and relative abundance of non-absorbing aliphatic moieties. ACS Paragon Plus Environment

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1. Introduction

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Natural organic matter (NOM) undergoes intense abiotic and microbial transformation in the

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environment. Its rate and depth depends on different environmental conditions including type and

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quantity of biomass, temperature, biological activity, humidity, and others.1 The progressive

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oxidation of peptides, lipids, and polysaccharides leads to accumulation of structurally diverse

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aromatics-rich compounds bearing both donor and acceptor groups, whose interactions give rise to a

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characteristic “long-wave tail” of the NOM absorption spectrum.2 The more extended system of

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interacting chromophores provides for the higher long-wave absorption, which has been intensively

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used in soil chemistry to characterize “humification degree” of soil organic matter with a ratio of

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absorbance values at 465 and 665 nm (E4/E6 ratio).4-6 Two different photophysical models are

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currently under discussion, which have been developed to describe characteristic optical properties

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of NOM: one of them considers NOM as a system of non-interacting chromophores, it will be

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referred to hereinafter as “superposition model”, whereas the other one implies charge-transfer

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interactions between NOM chromophores – “charge-transfer (CT) model”.7-9 The both models are

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widely used to predict optical properties of NOM, which largely impact aquatic photochemistry and

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microbial activity.10 Recently, new arguments have been set forth against the charge-transfer model.9

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However, they have not been backed up by the corresponding structural studies.

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Given that important prerequisite for charge-transfer interactions is a close spatial proximity

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of the donor and acceptor,11 it was of interest to find a structural parameter of NOM, which could be,

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on one side, related to spatial arrangement of donor and acceptor centers in supramolecular ensemble

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of NOM, and, on the other side, reasonably easy measurable. The major structural patterns of NOM

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include oxidation products of lipids, lignins, tannins, cellulose, and peptides.12 Hence, the major

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donating centers might be represented by lignin-derived phenolic units, whereas the major acceptor ACS Paragon Plus Environment


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centers might be tannin-derived quinones – or carboxyl enriched rings.13 Both types of groups are

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aromatic, and their spatial proximity may be ruled out by the presence of bulky substituents, e.g.,

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branched, multiple, or annealed aliphatic groups. This effect was studied in detail by Rathore et al.14

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who demonstrated that a group of three (1,3,5) ethyl substituents was sufficient for the complete

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steric inhibition of the face of a benzenoid (donor) chromophore for intermolecular association by a

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π–acceptor. The authors concluded that the difference of 0.9 Å between the separation of 3.6 Å in

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the CT complex [hexamethylbenzene, chloranil (CA)] and the predicted separation of 4.5 Å in

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[hexaethylbenzene, CA] could represent a “gray” area in which very weak, but visually (color) and

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spectrally (CT) observable association may be apparent.

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The other type of steric hindrance was recently envisioned by Chen et al15 for theoretic

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explanation of red shift absorption of eumelanin –skin pigment, which is also often considered as

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humic substances (HS) precursor.16,17 The authors reported that the excitonic interaction between

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molecules in aggregates lead to the appearance of new (shifted) spectral bands that is a classical

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effect in optical spectroscopy.15 However, this fact alone could not explain the exponential tail of

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absorption: simple superposition of melanin aggregates spectra did not yield NOM-like absorption

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spectrum. Chen et al. could achieve this effect by introducing the disorder into the system (i.e.

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Gaussian distribution of distance and angle between the aggregates of melanin molecules), which

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lead to the effective averaging of all possible shifted spectra and resulted in an overall exponentially

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fading absorption.15 In case of NOM this would mean that aside from dependence on the amount and

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quality of chromophoric groups (superposition model), the long-wave tailing of NOM spectra might

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be governed by the quality and quantity of non-absorbing units which induce disorder in the system

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of interacting chromophores (charge-transfer model). Of interest is that the both considered

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phenomena imply a particular role of aliphatic units, which are both bulky and non-absorbing, in

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imposing steric hindrance onto spatial arrangement of donor-acceptor pair which results in inhibition ACS Paragon Plus Environment

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of their charge-transfer interactions. At the same time detailed assessment of aliphatic building

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blocks was considered insignificant for describing UV/Vis behavior of NOM.

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In case of the NOM system, these non-absorbing and bulky units can be easily attributed to

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abundant aliphatic constituents such as materials derived from linear terpenoids (MDLT),18

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carboxyl-rich alicyclic moieties (CRAM),19 as well as by branched sidechains of proteins, waxes,

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suberins, and their associates.20-,22 Open chain terpenoids and proteins are much more amenable to

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biodegradation and photooxidation as compared to alicyclic and branched structures.18 Hence,

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progressive humification of biomass should be accompanied by an increase in contribution of

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oxygenated alicyclic structures and branched methyl groups on the account of linear structures.21

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This makes of particular diagnostic value the changes in aliphatic region of DOM.

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The method which is most sensitive to the presence of saturated aliphatic moieties in the

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NOM ensemble is 1H NMR spectroscopy. The main analytical challenge here comes from the

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presence of both labile (exchangeable) and backbone (non-exchangeable) hydrogens in molecular

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ensemble of NOM. The exchangeable protons are presented by O- and N-containing functional

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groups: carboxylic (COOH), phenolic (ArO-H), amine (N-H), hydroxyl (O-H) etc., whereas the

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backbone protons belong to non-substituted and functionalized methoxyl, methylene, methin groups,

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and to aromatic rings.19,23-25 The major aliphatic compartments are presented by modified terpenes

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such as the mentioned above MDLT18 and CRAM.19 In addition, side chains of peptides, O, and N-

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acetyl polysaccharides, carbohydrates, ether bridges of lignins, etc. contribute to aliphatic region.21,22

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As a result, the labile hydrogens of functionalized moieties can overlap with resonances of backbone

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hydrogens when aprotic solvents are used for dissolution.26,27 At the same time, when NaOD/D2O is

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used as a solvent, all exchangeable protons of NOM and HDO appear as a single coalescent peak in

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the range of 4.5–4.8 ppm due to fast (on the NMR time scale) exchange.28



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For deriving quantitative data on proton distribution over NOM ensemble, a whole variety of

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experimental techniques is described in the literature.23,25 This includes elimination of water from

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the NOM sample, a use of aprotic solvents (e.g., DMSO-d6), a use of H-D exchange, and proton

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downfield shift by addition of CD3COOD for enhancing resolution of 1H NMR spectrum with regard

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to detection of non-exchangeable protons.26-28 Another approach uses suppression of the solvent

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resonances in the NaOD/D2O solutions of NOM by selective presaturation of the HDO protons. 29

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Improved solvent suppression techniques were applied for 1H NMR measurements of NOM in

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D2O.30-33 In case of CD3OD (the solvent is widely used in 1H NMR studies of the solid phase

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extraction (SPE) isolates of HS), presaturation is used to attenuate residual water.34,35 For excluding

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residual water in case of DMSO-d6, thoroughly dried samples of NOM and HS were used.26,36

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For elucidating a relationship between optical properties and structural features of both

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aliphatic and aromatic constituents of the NOM ensemble, we have assembled a sample set

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including the lesser humified materials from the high-latitude rivers in the Russian Arctic dominated

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by the permafrost run-offs (the Kolyma River basin)37-39and the more humified materials from the

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temperate-latitude rivers and lakes. We have measured proton distributions over exchangeable and

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non-exchangeable structural moieties by acquiring triades of spectra in aprotic solvent (DMSO-d6)

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alone, in DMSO-d6 in the presence of CF3COOD, and in D2O. We related the obtained 1H NMR

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data to the optical properties measured with a use of UV-Vis spectroscopy. We backed up our

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conclusions using complementary technique - Fourier transform ion cyclotron resonance mass

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spectrometry (FTICR MS) for measuring molecular composition of the representative sample set.

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2 Experimental

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2.1. Sample set description

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The set of high latitude permafrost-impacted NOM samples was isolated from the thaws of Yedoma

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Ice Complex deposit and from the watersheds in the Kolyma River basin as a part of the “Polaris ACS Paragon Plus Environment

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Project” expedition (www.thepolarisproject.org) during 2010-2013. The Yedoma deposit was

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located in Duvanny Yar (DY),40 watersheds included different locations at the mainstream of the

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Kolyma River, its four tributaries as well as lakes and streams. This “arctic” subset included 16

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samples. The set of lower latitude samples (“non-arctic”) included four samples from the rivers and

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lakes located in Moscow region (Russia) as well as the samples of fulvic acid (FA) and dissolved

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organic matter (DOM) from the Suwannee River (SRFA and SRDOM, correspondingly) located in

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the Georgia State, USA, – the standard samples of IHSS. The sampling locations and full sample

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descriptions are shown in Fig. S1 and Table S1, respectively.

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2.2. Isolation of NOM samples

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For isolation of NOM from the Kolyma River basin watersheds, water was sampled by pre-cleaned

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HDPE bottles. In-line 0.45 µm filters (AquaPrep 600, Gelman Sciences) were used for water

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filtration. NOM isolation was carried out using Varian Bond Elut PPL cartridges (Mega Pack, 5 g),

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Amberlite XAD-8 and XAD-16 resin (Rohm and Haas Ltd.). The Amberlite XAD-16 resin was used

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as a feasible replacement to XAD-8 whose production is discontinued by the producer. Concentrated

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HCl (HPLC grade) and solid NaOH (reagent grade) were used for samples acidification and for

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preparing 0.1 M NaOH solution, respectively. Alkaline extracts were desalted on the cation

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exchanging resin Amberlite 120R in H-form (Rohm and Haas Ltd.). HPLC-grade methanol was used

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for elution of NOM from the PPL cartridges.

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Water samples (20-40 liters) were filtered through 0.45 µm and acidified with concentrated

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HCl until pH 2. Solid phase extraction (SPE) of NOM was conducted using Varian Bond Elut PPL

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cartridges as described elsewhere.42,43 In brief, the PPL cartridge was rinsed with methanol; an

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acidified water sample was discharged through the cartridge with a flow rate of 40 mL·min-1, the

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cartridge was dried, and stored in the dark at 4ºC. The NOM was eluted using methanol at a flow

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rate of 2 mL·min-1. The obtained extracts were dried on rotor evaporator. Column extraction of ACS Paragon Plus Environment


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NOM using XAD-8 was conducted according to the standard IHSS protocol.44 In brief, the filtered

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water sample was acidified to pH 2 and discharged through the column packed with XAD-8 resin

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(2x20 cm). The resin was eluted with 0.1 M NaOH. The alkaline extract was immediately passed

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through the cation-exchanging resin in H-form. The desalted samples were rotor-evaporated to

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dryness. The same protocol was applied to XAD-16 resin. All dry samples were stored at –20ºC in

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the dark. All procedures are described in detail by Perminova et al.43 All sources of NOM samples,

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the sorbents used for their isolation, and the attributions are listed in Table S1.

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2.2. 1H NMR spectroscopy

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Solvents and Reagents. DMSO-d6 had isotopic purity of 99.95 % (water content ≤ 0.02 %, Merck,

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Darmstadt). Deuterated water (D2O, 99.95% D) and 40 % NaOD in D2O (99+% D) were purchased

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from Aldrich (Milwaukee, WI). NaOD was used for facilitation of dissolution of some samples. All

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reagents and solvents were used as received from the suppliers.

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1

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(Bruker, Germany) operating at 400 MHz 1H frequency. For measurements in DMSO-d6, the

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samples were prepared as described by Kovalevskii et al.26 In brief, a weight of 15 mg of NOM

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sample and 0.75 mL of anhydrous DMSO-d6 sealed in an ampoule were placed into a 5 mm NMR

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tube and 13 mm pyrex tube, respectively, which were adjusted to each other as a Schlenk-type

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apparatus. The apparatus was attached to a vacuum line and evacuated under high vacuum (< 5×10-2

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mm Hg) for 6 hours to remove hygroscopic water from the NOM sample. The apparatus was

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detached from the vacuum line and shaken to break DMSO-d6 ampoule and to drain the solvent into

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an NMR tube with the dried NOM sample. The NMR tube was sealed and used for spectrum

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acquisition. Then the tube was re-opened, added with 20 to 40 µL of CF3COOD and the spectrum

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was reacquired. For D2O measurements, a weight of 15 mg NOM was dissolved in 0.6 mL of D2O.

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All 1H NMR spectra were acquired in a 5 mm tube using 90 excitation pulses (acquisition time 3.2 s,

H NMR measurements. 1H NMR spectra were acquired using an Avance-400 NMR spectrometer


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relaxation delay 2 s, ~100 scans). Fourier transformation, phase correction and integration were

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performed using MestReC software (Mestrelab Research, USA).

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The integration of spectra was conducted using the following assignments (ppm): 0-1.15 ppm

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(CH3 – protons), 0.–2.05 – protons of alkyl chains (CHn – protons); 2.05–3.2 – protons of carbon

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located in α-position to carboxyl group or to aromatic ring (CHα – protons) (X-CαH, X: COOH,

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COOR, CAr), it might also carry resonances of amines and other functional groups; 3.2–6.0 – protons

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of alkoxy and aliphatic hydroxyl groups, amines and amides (CHnO(N), CHn-O(N)H); 6.0–7.0 –

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olephins, peptides, aminoacids, 7.0 -10.0 – aromatic and phenolic protons (CArH and CArOH; where

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CAr corresponds to aromatic carbon atoms); 10.0–18.0 – protons of carboxyls (COOH).26,31,45

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The spectral intensity of residual H atoms of DMSO-d6 at 2.5 ppm was subtracted from the

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spectral integral of CHα- protons by using the NOM signature at δ H ~ 2.5 ppm as a continual

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background. The spectral intensity of HDO peak was calculated as an integral from 4 to 5.5 ppm and

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subtracted from the total integral in the range from 0 to 18 ppm. The values of CHn and CHα

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integrals were used for calculation of CHn/CHα ratio.

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2.4. UV-Vis spectroscopic measurements and data treatment

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A weight of the NOM sample was dissolved in 0.03 M phosphate buffer at pH 6.8 to yield

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concentration of 20 mg·L-1. UV-Vis spectra were recorded using Cary-50 spectrophotometer (Varian

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Inc.) in the range from 200 to 800 nm. The recorded data were used to calculate E4/E6 ratio as a ratio

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of absorbance values at 465 and 665 nm. This ratio is traditionally used to estimate humification

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degree of organic matter.3,4 Another parameter calculated from the UV-Vis data is similar to the

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chromaticity coefficient of HS.1 For this purpose the long wave (>500 nm) part of a UV-Vis

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spectrum was approximated by eq (1):

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A(λ) = A0·exp(-λ/Λ) + a,


(1)


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where: A is the optical density; λ is the wavelength (nm); Λ (nm) is the slope of absorption in

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semilogarithmic scale, and a is the offset connected to nonideal measurements of absorption spectra.

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Eq 1 is based on logarithmic approximation of the long-wave tail of the absorbance spectra

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described by the Urbach rule.46 The calculated slope value (Λ ) was used for evaluation of long-wave

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tail of NOM.

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2.5 Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS)

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FTICR mass-spectra were recorded using a hybrid LTQ FT Ultra (Thermo Electron Corp., Bremen,

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Germany) mass-spectrometer equipped with a 7T superconducting magnet. Negative ions were

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produced by an IonMax Electrospray ion source (Thermo Electron Corp., Bremen, Germany).

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Needle voltage was -3kV. The direct infusion rate was 1 µL·min-1 Temperature of the desolvating

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capillary was 200 oC. Broadband MS spectra (m/z 200-1000) were acquired in the FT ICR with a

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resolution (R) of 400000 at m/z 400 by a consecutive summation of 300 scans. Internal calibration

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was systematically conducted using the known peak series of NOM and the internal carboxylated

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styrene calibrant.47,48 The NOM samples were dissolved in methanol (HPLC grade) at concentration

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of 1 mg·mL-1. The data treatment is described in the Supporting Information,

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3. Results and discussion

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3.1. Solution state 1H NMR spectroscopy and UV-Vis on the NOM samples used in this study

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1

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of CF3COOD (TFA-d), and D2O. This was to observe full distribution of both backbone and

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exchangeable protons in the NOM samples used in this study. The spectrum in DMSO-d6 is a

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superposition of both exchangeable and backbone protons, whereas the spectra in DMSO-d6/TFA-d

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and D2O contain only backbone protons and differ in the position of residual solvent protons: at 2.5

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ppm (DMSO-d6) and 4.7 ppm (HDO). Acquisition of all three spectra enables normalization of

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spectral intensity and calculation of full proton distribution. The “triads” of the obtained 1H NMR

H NMR spectra were acquired using different NMR solvents – DMSO-d6, DMSO-d6 in the presence


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spectra typical for the “arctic” versus “non-arctic” NOM samples used in this study as well as for the

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SPE-PPL versus the XAD-isolates from the same source are shown in Fig. 1. The “triads” were

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acquired for 17 out of 27 samples used in this study. They are shown in full in Fig. S2 of the SI.

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A

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C

B

D

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Figure 1. The typical triads of 1H NMR spectra acquired in DMSO-d6, DMSO-d6/TFA-d, and D2O

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(highlighted in dark-purple, green, and blue, respectively) for the arctic NOM (Yedoma thaw, AHF-

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DYP 11) (A) versus the non-arctic NOM (the Suwannee River DOM, SRDOM) (B), and the 1H

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NMR spectra of the PPL isolate – AHF-LSP-12 (highlighted in blue) versus the XAD-8-isolate –

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AHF-LSX12 (highlighted in orange) from the same source – the Sion Lake (Moscow Region)

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acquired in D2O (C) and DMSO-d6 (D).



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The major difference among the spectra in the three solvents used can be seen in the region

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of backbone aliphatics (0-3.2 ppm) occupied by the most “rigid” or sterically encumbered building

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blocks of NOM, nominally, by MDLT and CRAM.18,19 In case of the typical “arctic” sample (Fig.

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1A) it is largely dominated by non-functionalized alkyl protons of MDLT and other lipids signaling

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from 0 to 2.05 ppm (CHn). Their contribution exceeds substantially intensity of downshifted CHα

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protons usually attributed to CRAM (2.05 -3.2 ppm). In the shown representative of the “non-arctic”

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samples (Fig. 1B), the intensity of CHn protons is drastically reduced (it becomes comparable with

252

CHα) – mostly, on the account of protons of carbohydrates and peptides signaling from 3.2 to 6

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ppm. These trends might be indicative of the higher content of the least altered aliphatic units into

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the structure of arctic versus non-arctic NOM samples, which is consistent with the differences in

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their genesis: the low temperatures of the permafrost-dominated Kolyma region preclude rapid

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decomposition of organic matter, whereas the elevated temperatures of temperate and other non-

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arctic regions accelerate transformation processes of organic matter. However, except for the genetic

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features, there is a purely analytical factor, which could also impact greatly the structural

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composition of the NOM isolates: this is sorbent selectivity, which was considered in detail in our

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previous publication.43 Its impact on structural features of the NOM isolates extracted from the same

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source with a use of different sorbents is shown in Figs. 1C,D on the example of the NOM samples

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extracted from the same source – the Sion Lake located in the temperate latitudes of the Moscow

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region – using the SPE PPL cartridge and the Amberlite XAD-8 resin. The 1H NMR spectra for the

264

both samples were acquired in two different solvents – D2O and DMSO-d6, which are shown in Figs.

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1C and 1D, respectively. It can be seen that the PPL isolate is characterized with the higher content

266

of aliphatic units as compared to the XAD-8 isolate: the latter, on contrary, is enriched with the

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aromatic units. This is in line with the trends which were revealed in our previous studies.43 Hence,

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the particular caution should be exercised upon comparing the NOM isolates, which were obtained ACS Paragon Plus Environment

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with a use of different sorbents. This topic was studied and described in detail by Li et al.35 The

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quantitative assessment of the proton distribution in all NOM samples used in this study is shown in

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Fig. 2 for DMSO-d6 and D2O, the corresponding numeric values are given in Tables S2-S4 (SI). The

272

full size 1H NMR spectra in all three NMR solvents are shown in Figs. S3-S5 in the SI.

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A

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Figure 2. 1H NMR spectra acquired in DMSO-d6 (left panel, A) and D2O (right panel, B) with the

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color-coded integral intervals, in ppm: 0-1.15 (yellow), 0-2.05 (light brown), 2.05-3.2 (violet, 3.2 -6

276

(purple), 6-7 (turquoise), 7-10 (dark green), 10-18 (light green).

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As follows from Fig. 2, the largest amount of hydrogen in the NOM samples under study belongs to

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non-functionalized aliphatic building blocks (e.g., MDLT),51 which are highlighted in brown. Their

279

content reaches up to 40% of the total (labile and backbone) hydrogen (Fig. 2A), and the consistently

280

higher values are observed in the arctic (30-40%) versus non-arctic (20 to 30%) isolates. The

281

respective values account for 40-50% and 30-35% of non-exchangeable hydrogen as follows from


B


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D2O and TFA spectra (Fig, 2B and Table S4, respectively). Given the high relevance of branched

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aliphatic units for the steric hindrance effect, we estimated the amount of methyl groups (shown in

284

yellow in Fig. 2) in the samples under study. It accounted for about 15% of the total hydrogen for the

285

arctic samples and did not exceed 10 % in the non-arctic ones. The corresponding ratios of CH3 to

286

(CH2+CH) and (CH2+CH+CHα) protons varied from 1:(1.6 – 1.8) and 1:(3.5-3.7), respectively, for

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arctic NOM, to 1:(2.1-2.3) and 1:(4.5-5.5) for SRDOM and other non-arctic NOM. The obtained

288

values are indicative of rather extended methin-methylene core of the samples under study, in

289

particular, in those of arctic origin. Its branching degree is lower than in linear terpenoids (e.g.

290

squalene) or bacterial hopanoids, where the corresponding ratios could be estimated from 1:1.5 to

291

1:1.49 This could be connected to substantial contribution of non-branched lipids such as waxes and

292

fatty acids into terpenoid-like material, maybe, in the form of associates of hopanoids and cell-wall

293

lipids49 or with other methylene-rich bacterial compounds, e.g. branched glycerol dialkyl glycerol

294

tetraethers (brGDGT), which are important biomarkers of the high latitude OM.50

295

The content of CHα - protons varied not so remarkably – between 20 and 25 % of the total

296

hydrogen for all isolates and reached its minimum value (15%) for the Yedoma thaws (AHF-DYP10

297

and –DYP11). These protons are usually assigned to CRAM,19 which are produced by photo- and

298

microbial oxidation of terpenoids.18,21 The obtained trend looks rather consistent with the samples

299

origin: the Yedoma NOM was depleted with these highly processed NOM constituents. It is also in

300

line with the higher content of carbohydrate/peptide units (from 3.2. to 6 ppm) in these samples as

301

compared to the other arctic samples confirming their least degraded character.39-41 The CHα protons

302

also reflect amount of CH-substituted aromatic rings in the system, which might explain the highest

303

intensity of this interval for the non-arctic samples enriched with aromatics. This corroborates the

304

higher content of aromatic and COOH protons in these samples signaling from 6 to 14 ppm.


Page 15 of 33


305

Summarizing the obtained results, the conclusion can be made that the “arctic” part of the

306

assembled NOM sample set was characterized with much higher contribution of methylene-enriched

307

non-functionalized aliphatic units usually assigned to MDLT compartments as compared to the non-

308

arctic isolates. The content of these CHn or “MDLT” protons in the arctic samples was up to a factor

309

of two higher than the content of CHα protons of CRAM- and aromatic constituents; in the non-

310

arctic samples it dropped to one. This motivated us for using a ratio of CHn to CHα protons (it can

311

be also considered as a proxy for MDLT to CRAM ratio) as a parameter reflecting the prevalence of

312

rigid non-oxidized hydrophobic core over the more polar, C-H acidic aliphatic units. Given that

313

these are non-functionalized rigid alicyclic moieties and branched chain proteins which contribute

314

the most into the amount of CHn protons, these very moieties might induce an effect of steric

315

hindrance by causing distortions in planar orientation needed for charge-transfer interactions of the

316

aromatic rings. In this case, a ratio of CHn to CHα protons might serve as a structural parameter

317

indicative of the size and “bulkiness” of the aliphatic part of the supramolecular ensemble of NOM

318

reflecting its feasibility for charge-transfer interactions and their contribution into the optical

319

properties of NOM. Hence, the NOM samples with the higher CHn/CHα ratio (e.g., the arctic subset

320

in case of this study) will be characterized with least pronounced charge-transfer interactions,

321

whereas the samples with the low CHn/CHα ratio (e.g., the non-arctic subset) will be characterized

322

with much higher contribution of charge-transfer interactions into their optical properties. On the

323

other side, this very ratio might serve as an NMR equivalent of humification degree of the NOM

324

ensemble while it reflects contribution of least transformed extended aliphatic structures versus

325

residual aliphatic structures adjacent to aromatic group of carboxyl resulting from progressive

326

oxidation of biomass during its transformation. For proving our working hypothesis on the

327

relationship between structural arrangement of aliphatics and its feasibility to interfere with charge-



Page 16 of 33

328

transfer interactions in the NOM ensemble, we have measured the optic properties of the NOM set

329

used in this study.

330

3.2. Structure – optical properties relationships as deduced from 1H NMR and UV-Vis data

331

UV-Vis spectra of the samples under study were typical for NOM showing monotonous decrease of

332

absorbance along with an increase in a wavelength (Fig. 3A). They were characterized using the

333

E4/E6 value,3,4 which reflects steepness of the “long-wave tail” of the NOM spectrum, and the Λ

334

parameter, which is a reverse value of the slope of red edge absorption plotted in semilogarithmic

335

scale (Fig. 3A). The obtained E4/E6 and Λ values along with CHn/CHα ratio are given in Table 1.

336 337

Figure 3. UV-Vis spectra of NOM in semilogarithmic scale show good linearity in the range from

338

450 up to 700 nm, the red lines correspond to fits of absorption spectra using eq (1) (A);

339

visual representation of the Spearman correlation coefficient for the 1H NMR data and both optical

340

parameters used in this study: the E4/E6 ratio and the Λ parameter (n=32, p = 0.95) (B)

341


Page 17 of 33


342

Table 1. The calculated CHn/CHα ratio and optical parameters characterizing long wave absorption

343

of the NOM samples used in this study Sample

Description of NOM source

Sorbent

the Yedoma deposit thaw – “arctic” NOM

CHn/CHα D2O

DMSOd6

E4/E6

Λ nm

AHF-DYP-10

Duvanny Yar (2010)

PPL

2.0

2.4

15.2

75

AHF-DYP-11

Duvanny Yar (2011)

PPL

2.1

2.0

13.7

70

AHF-DYP-13

Duvanny Yar (2013)

PPL

1.6

1.6

16.1

75

the Kolyma River basin watersheds (‘arctic” NOM) AHF-AOP-10 KR* estuary (2010) PPL

1.8

1.8

19.0

69

AHF-FPP-10

Flood plane stream (2010)

PPL

1.8

2.4

23.1

66

AHF-LTdP-10

Lake Tubdispanser (2010)

PPL

1.8

1.8

20.1

67

AHF-RK5P-10

KR near Chersky (2010)

PPL

-

1.8

26.1

70

AHF-RK6P-10

KR at Omolon River (2010)

PPL

1.9

2.0

16.3

73

AHF-RKP-12

KR mainstream (2012)

PPL

1.9

-

17.1

66

AHF-RKX8-11

KR near Chersky (2011)

XAD-8

-

1.4

15.1

83

AHF-RMAP-10

Malyi Anyui River (2010)

PPL

1.9

-

17.4

73

AHF-RMAX16-12

Malyi Anyui River (2012)

XAD-16

1.3

-

10.4

86

AHF-RMdP-10

Medvedka River (2010)

PPL

1.9

1.6

17.7

72

AHF-RPP-10

Panteleikha River (2010)

PPL

1.8

1.7

14.7

76

AHF-RPP-11


PPL

1.8

1.8

12.0

72

AHF-RPX8-11


XAD-8

1.8

1.7

10.0

83

AHF-RSkP-13

Sukharnaya River (2013)

PPL

1.8

-

16.2

66

the lower latitude watersheds (“non-arctic” NOM)

344

AHF-RIsX8-04

Istra River, Moscow, Russia

XAD-8

1.6

-

12.6

81

AHF-RMX8-97

Moscow River, Russia

XAD-8

1.6

-

9.6

88

AHF-LSP-12

Sion Lake, Moscow, Russia

PPL

1.5

1.5

11.0

87

AHF-LSX8-12

Sion Lake, Moscow, Russia

XAD-8

1.0

1.0

10.0

104

SRDOM

Suwannee River, IHSS

RO**

1.4

0.9

11.8

84

SRFA

Suwannee River, IHSS

XAD-8

1.1

1.1

12.7

79

*KR stands for the Kolyma River, **RO stands for reverse osmosis.

345 ACS Paragon Plus Environment


Page 18 of 33

346

As it can be deduced from Table 1, the highest E4/E6 values were characteristic for all PPL isolates

347

of arctic NOM reaching the maximum value of 26.1, whereas much lower values ranging from 9.6 to

348

12.7 were observed for the non-arctic samples (Table 1). In general, the obtained values of E4/E6

349

estimates corroborated well the reported ones for the NOM samples from different watersheds.

350

O’Driscoll et al. studied NOM from lakes and obtained E4/E6 ratios in the range from 4.5 to 16.52

351

Chin et al. reported the E4/E6 values for fulvic acids isolated from different rivers in the range from

352

9.7 (the Yakima River) to 21 (the Suwannee River).53 At the same time, the authors stated that the

353

optical density at 665 nm for the NOM samples could be quite low bringing about significant errors

354

in E4/E6 estimation.54 This was the same reason why we have used the Λ parameter, whose

355

measurement is based on approximation of the whole long-wave range of absorption spectra of

356

NOM. The corresponding Λ values varied from 66 to 91 for the arctic NOM used in this study, and

357

from 81 to 104 for the NOM samples isolated from the lower latitude watersheds (Table 1).

358

The revealed trends in optical properties indicated the smaller long-wave absorption of the

359

arctic versus non arctic NOM isolates used in this study. The obtained data set was used for

360

correlation analysis with the three sets of 1H NMR data calculated from the spectra measured in

361

D2O, DMSO-d6, and DMSO-d6/TFA-d. The results of correlation analysis are shown in Fig. 3B. The

362

Spearman correlation was applied due to the different sizes of the sample subsets. For all three 1H

363

NMR sets, the strongest statistically significant correlation with the Λ parameter (P=95%) was

364

observed for CHn/CHα (D2O –0.65, DMSO-d6 –0.82, DMSO-d6/TFA-d –0.81); CHn (D2O –0,6;

365

DMSO-d6 –0.83, DMSO-d6/TFA-d – 0.87), and HAr (D2O + 0,61; DMSO-d6 +0.79, DMSO-d6/TFA-

366

d +0.93). There was no statistically significant correlation observed for COOH. The similar

367

correlation trends were observed for the E4/E6 parameter. Given the specific feature of the NOM

368

sample set used in our study that the “arctic” samples were isolated mostly with a use of the SPE

369

PPL cartridges, whereas the”non-arctic” samples were isolated mostly using the Amberlite XAD ACS Paragon Plus Environment

Page 19 of 33


370

resins, it was of interest to validate the obtained correlation relationship for the subset of samples

371

isolated with a use of the same isolation technique. For this purpose we conducted the same

372

correlation analysis for the PPL subset composed of 16 samples for all three 1H NMR data sets:

373

D2O, DMSO-d6, DMSO-d6/TFA-d (given in Tables S5-S6). The results of correlation analysis

374

(shown in Tables S7-S8) confirmed the trends obtained for the full data set: the closest correlation

375

with the Λ parameter (P=95%) was observed for CHn/CHα (D2O –-0.652, DMSO-d6 –0.639,

376

DMSO-d6/TFA-d –0.777).

377

In terms of charge-transfer interactions, the obtained relationships demonstrate that

378

enrichment of the NOM samples with the non-functionalized aliphatics weakens charge-transfer

379

interactions in the NOM ensemble, which is reflected in less pronounced long-wave tailing. The

380

obtained results support our initial hypothesis that bulky (or extended) aliphatic moieties might

381

prevent proximity of aromatic donor and acceptor centers which is necessary for formation of

382

charge-transfer complexes.

383

3.3. Molecular composition of the NOM samples under study and its relation to 1H NMR

384

structures and optical properties

385

For further exploration of the revealed structure-optics relationships, we have undertaken in depth

386

studies on molecular composition of the NOM samples used in this study using complementary to

387

1

388

FTICR MS including both arctic and non-arctic subsets. Fig 4 represents Van Krevelen (VK)

389

diagrams for the arctic (left panel) and non-arctic (right panel) NOM samples used in this study. All

390

four samples shown in Fig. 4 were characterized by the dominant contribution of unsaturated CHO

391

compounds with O/C≈0.5, which are considered as the most conservative part of NOM8 and could

392

be related to CRAM.19

H NMR spectroscopy technique – FTICR MS. Nine out of 22 NOM samples were analyzed using



Page 20 of 33

393 394

Fig. 4. Van Krevelen diagrams for A) AHF-DYP10, B) AHF-AOP10, C) AHF-LSP12, D) SRDOM.

395

Dot size represents relative abundance of the corresponding peak in FTICR mass-spectra.

396

Attribution of the VK regions is according to55,56

397

The Yedoma-derived sample (AHF-DYP10) was characterized with the highest contribution

398

of aliphatic and peptide components (MDLT), whereas non-arctic sample SRDOM was enriched

399

with the most oxidized species (O/C>0.6) characteristic of hydrolyzable tannins. The latter were

400

much less abundant in the arctic NOM samples. These trends become even more obvious upon

401

segregation of the VK diagram field into the molecular classes using aromaticity index:55,56 the

402

major contribution was observed for the unsaturated species with H/C1.5) was higher in the arctic versus and non arctic NOM samples. For

404

relating the found trends in molecular composition to 1H NMR data and optical parameters, we have

405

identified common formulas present in all nine NOM samples (~650 out of ~4000 total) and used

406

their intensities for further correlation analysis57 (Fig. 5). ACS Paragon Plus Environment

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407 408

Fig. 5. Correlation of the relative abundance of the common formulas of nine NOM samples

409

measured using FTICR MS with 1H NMR (DMSO-d6) and optical parameters. The colors designate

410

a positive (red) or negative (blue) value of Spearman correlation coefficient (P>95%).

411

It can be seen that 1H NMR data produce rather meaningful correlations with the abundance trends

412

in the molecular constituents of the NOM set used in this study. Positive significant correlations are

413

observed both for the CHn and CHα parameters with the molecular constituents located in the

414

[unsaturated and CRAM] region for the molecules with O/C0.5 region [aromatic and polyphenols] and negatively - to the aliphatic region. The content

418

of COOH was directly related to the population density of carboxyl-rich hydrolyzable tannin region.

419

Of particular interest are correlations with the structural and optical parameters proposed in our

420

study as characteristics of long-wave absorption: CHn/CHα and Λ, respectively. CHn/CHα directly

421

correlated with rather narrow suite of formulas located in the unsaturated region with O/C~0.5, and

422

was inversely proportional to abundance of a broad suite of formulas in the polyphenol region. The



Page 22 of 33

423

similar trends were observed both for Λ and E4/E6 parameters: they were most sensitive to the

424

abundance of MDLT and aromatic regions.

425

The observed inverse relationship between the Λ parameter and the abundance of aliphatic

426

and low-oxidized unsaturated compounds might be seen as supportive of our working hypothesis on

427

the steric hindrance of charge-transfer interactions. Indeed, unsaturated compounds with H/C> 1 and

428

low O/C ratios can be assigned both to aromatic structures with aliphatic side-chains and to CRAM,

429

which are bulky alicyclic moieties and cause an increase in the distance between interacting aromatic

430

fluorophores. The direct correlation between Λ and the content of highly oxidized unsaturated

431

compounds, in turn, might indicate importance of the relative abundance of donors and acceptors in

432

the molecular ensemble of NOM for its optical properties. This is in line with the reported data on

433

reduction of carbonyl groups of DOM by sodium borohydride causing a loss of visible absorption

434

and inducing blue-shift of fluorescence spectra.58 The observed effects were explained as a

435

significant weakening of charge-transfer interactions. The similar trends might be deduced from the

436

data obtained in our study: firstly, the content of oxidized species– the major electron acceptors in

437

the NOM ensemble was significantly correlated with the Λ value (Table S9), which is proportional

438

to long-wave tailing caused by charge-transfer interactions; secondly, an increase in the CHn/CHα

439

ratio was directly proportional to the content of aliphatic and low-oxidized unsaturated molecules,

440

which cause a decrease in the Λ value. Of interest is also a direct relationship between the value of

441

CHα integrals in the 1H NMR spectra and the abundance of highly-oxidized unsaturated molecules

442

in the FTICR MS spectra (Table S9, Fig. S6), which might be indicative of oxidative transformation

443

of methyl groups during aromatization of terpene residues. In general, the obtained data provides

444

additional arguments in favor of steric mechanism of the impact which aliphatic compounds

445

(terpenoids and lipids) and substituents might impose on the visible absorbance of NOM.


Page 23 of 33


446

3.4. Rationale on the role of aliphatic moieties in charge-transfer interactions in the

447

supramolecular ensemble of NOM

448

In the context of the obtained results and in line with the both previously discussed mechanisms of

449

the steric hindrance,14,15 it can be suggested that the prevalence of non-functionalized aliphatic

450

molecules/fragments (e.g. MDLTs) in the molecular ensemble of NOM might change distribution of

451

the relative positions of major chromophoric groups by shifting the respective intermolecular

452

distances to the values higher than 3.6 Å (Fig. 6). This might lead to a decrease in the strength of

453

charge-transfer interactions resulting in the lower absorption in the red edge of the spectrum. Increased fraction of aliphatic moieties

Aliphatic moieties

r r NOM NOM Chromophoric groups

454

Distance between chromophoric groups (r)

455

Figure 6. Distribution of distances between the chromophoric groups depends on the ratio of

456

aliphatic/aromatic moieties (CHn/CHα ratio) in the NOM supramolecular ensemble.

457 458

This looks overall plausible given that the MDLTs are mostly represented by sterically

459

encumbered modified linear and alicyclic terpenoids (e.g., squalene, sterans, hopanoids), other low-

460

functionalized lipids (e.g. waxes, fatty acids, branched glycerol dialkyl glycerol tetraethers),

461

branched side-chain peptides. Due to extended sp3 backbone these compounds are essentially

462

hydrophobic and CH2-chains might produce crystalline-like domains as it was previously envisioned



Page 24 of 33

463

for soil HS.59 Mere hydrophobic interactions of these large aliphatic domains with aromatic rings

464

might increase the distance between the chromophoric groups to more than 3.6 Å leading to partial

465

inhibition of the charge-transfer interactions or to their complete extinction if the separation exceeds

466

4.5 Å.14 Progressive oxidation of the constituents of these hydrophobic domains (e.g., CRAM

467

formation) will increase solubility and reduce their size leading to more polar, functionalized and

468

labile (much less rigid) molecular ensemble of NOM, which will facilitate spatial proximity needed

469

for stronger charge-transfer interactions. The proposed conceptual model is in good agreement with

470

the existing data on NOM interactions with hydrophobic compounds with extended π-system such as

471

polycyclic aromatic hydrocarbons.60,61 It might also have direct implications for predicting

472

antioxidant and “pro-oxidant” activities of NOM and HS from the proton distribution in their

473

molecular ensemble.

474 475

Acknowledgements

476

Authors would like to acknowledge the outstanding contribution of the anonymous ES&T reviewer

477

whose insightful comments and suggestions enabled substantial improvement of the initial version

478

of the manuscript. AK, AZ, VL, NK and IP appreciate support of the Russian Science Foundation:

479

grant # 16-14-00167 (1H NMR spectroscopic analysis of the NOM samples and data interpretation),

480

AZ and EN – RSF grant #14-24-00114 (high-resolution mass spectrometry studies), AK, AZ, NK,

481

ID and IP appreciate support of the Russian Foundation for Basic Research (grant # 16-04-01753 -

482

isolation and primary characterization of the NOM samples), ES and IP – grant RFBR 15-05-09284

483

(optical studies of the DOM samples), ID, VL, EB and MH appreciate support of the NSF Polaris

484

project (sampling campaign in the Kolyma River basin). ES was supported by the Young

485

Investigator Research grant from the International Humic Substances Society. Authors are grateful to

486

developers of the open source Matplotlib software. ACS Paragon Plus Environment

Page 25 of 33


487 488

The authors declare no conflict of interests

489

Supporting Information (SI) available for this manuscript contains extended information on the

490

samples (Fig. S1,Table S1), 1H NMR spectra and spectral integrals (Figs. 2-5), Tables S2-8,

491

description of FTICR MS data treatment and results (Table S9, Fig. S6). This material is available

492

free of charge via the Internet at http://pubs.acs.org.

493

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494

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495

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4. Y. Chen, N. Senesi and M. Schnitzer, Information provided on humic substances by E4/E6

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271x114mm (96 x 96 DPI)


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