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Soil organic matter in its native state: unravelling the most complex biomaterial on Earth Hussain Masoom, Denis Courtier-Murias, Hashim Farooq, Ronald Soong, Brian P. Kelleher, Chao Zhang, Werner E. Maas, Michael Fey, Rajeev Kumar, Martine Monette, Henry J Stronks, Myrna J Simpson, and Andre J Simpson Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03410 • Publication Date (Web): 19 Jan 2016 Downloaded from http://pubs.acs.org on January 20, 2016

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Environmental Science & Technology

FOR SUBMISSION TO ENVIRONMENTAL SCIENCE AND TECHNOLOGY

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Soil organic matter in its native state: unravelling the most complex biomaterial on Earth

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Hussain Masoom1, Denis Courtier-Murias1, Hashim Farooq1, Ronald Soong1, Brian P. Kelleher2,

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Chao Zhang1, Werner E. Maas3, Michael Fey3, Rajeev Kumar4, Martine Monette4, Henry J.

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Stronks4, Myrna J. Simpson1, and André J. Simpson1*

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1. Department of Chemistry, University of Toronto, Toronto, ON, Canada, M1C 1A4

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2. School of Chemical Sciences, Dublin City University, Dublin, Ireland

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3. Bruker BioSpin Corp., Billerica, Massachusetts, USA, 01821-3991

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4. Bruker BioSpin Canada, Milton, ON, Canada, L9T 1Y6

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*Corresponding Author Information:

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André J. Simpson Department of Chemistry University of Toronto Scarborough 1265 Military Trail Toronto, Ontario. Canada, M1C 1A4 Telephone: 1+(416)-287-7547

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Email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT (199/200 words)

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Since the isolation of soil organic matter in 1786, tens of thousands of publications have

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searched for its structure. Nuclear magnetic resonance (NMR) spectroscopy has played a critical

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role in defining soil organic matter but traditional approaches remove key information such as

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the distribution of components at the soil-water interface and conformational information.

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Here a novel form of NMR with capabilities to study all physical phases termed Comprehensive

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Multiphase NMR, is applied to analyze soil in its natural swollen-state. The key structural

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components in soil organic matter are identified to be largely composed of macromolecular

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inputs from degrading biomass. Polar lipid heads and carbohydrates dominate the soil-water

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interface while lignin and microbes are arranged in a more hydrophobic interior. Lignin domains

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cannot be penetrated by aqueous solvents even at extreme pH indicating they are the most

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hydrophobic environment in soil and are ideal for sequestering hydrophobic contaminants.

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Here, for the first time, a complete range of physical states of a whole soil can be studied. This

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provides a more detailed understanding of soil organic matter at the molecular level itself key

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to develop the most efficient soil remediation and agricultural techniques, and better predict

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carbon sequestration and climate change.



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INTRODUCTION Soil is the foundation of all life on Earth. It acts as a habitat, a nutrient source, a carbon

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sink and mediator, a regulating body for water flow, a filter, a platform for infrastructure, and a

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source for minerals and resources.1 All living things rely on soil and its degrading health has a

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far reaching impact. With a warming planet comes the possibility of a release of carbon into the

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atmosphere from the largest active organic pool on the planet, further exacerbating global

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warming.2-4 Soil erosion also contributes to global warming and food instability.4, 5 Less soil

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equates to less area for agriculture. With agricultural, industrial and household processes

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comes the release of contaminants which irreversibly bind to hydrophobic components in soil

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organic matter (SOM). Contaminant interactions with soil are poorly understood because of our

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lack of understanding of soil structure leading to inefficient remediation strategies.6-8

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Remediating sites in the USA and the European Union are estimated to cost 1.4 trillion Euros.9

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All of these processes are defined by a plethora of molecular-level interactions within the soil

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itself that define its chemistry, reactivity and kinetics. Therefore, a deeper understanding of soil

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structure is desperately required.

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Soil has been reported as the “most complex biomaterial on earth” and it is widely

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accepted that the structural complexity of SOM and lack of understanding thereof is a major

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impediment to progression in the field.10 Traditionally SOM was thought to be comprised

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predominately of a unique category of cross-linked structures termed humic substances.

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However, more recent work has shown that extracted SOM is better described as a complex

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mixture of plant and microbial inputs at various states of decay present at the time of

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extraction.11, 12 In 2011 Schmidt et al. published a seminal review highlighting that a wide range


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of ecosystem properties will also define the persistent organic residues in soil. As such,

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understanding the structure, water accessibility, conformation, interfaces, and reactivity of

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SOM with its surrounding environment and in its natural swollen state is now more imperative

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than ever. One major impediment to studying soil structure has been the lack of instrumentation

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to study complex environmental samples in their natural state.13 This instrumentation must be

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able to provide the molecular-level resolution required to identify and quantify the components

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present and their associations. Nuclear magnetic resonance (NMR) spectroscopy is a powerful

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tool to study complex environmental samples and their interactions at a molecular level.14 Traditionally, however, NMR spectroscopy has developed largely as two separate fields,

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one dealing with solid crystalline structures and the other studying soluble compounds. Soil

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contains a broad continuum of structures ranging from crystalline solids to solution-like

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compounds thus instrumentation to study all of these components is required. Solid-state NMR analysis requires the least amount of sample preparation, however 1H–

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be obtained from 1H NMR resulting in the less sensitive 13C nucleus most commonly being used

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for detection.14, 15 While solution-state NMR can provide very high resolution fingerprints of

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SOM, the technique is restricted to soluble components only.

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H dipolar interactions in the solid state are considerable and reduce the information that can

The situation was improved in 1999 with the advent of high resolution magic angle

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spinning (HR-MAS) a technique that can study both soluble and semi-solid components.16 HR-

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MAS NMR on whole soils yields high resolution spectra with the ability for multidimensional



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analysis, and has been applied to study whole soil interactions with contaminants at the soil-

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water interface.17 However, HR-MAS probes do not have the radio frequency power handling

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capacities to study the solid component in soils which play a key role in long term contaminant

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sequestration and soil stabilization.18-20

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In their natural state, soils exhibit a continuum of phases with everything from

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crystalline solids to true solutions being present. In this manuscript we employ the use of novel

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Comprehensive Multiphase (CMP) NMR which combines all the electronics from solution-state,

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semi-solid and solid-state NMR into a single NMR probe.21 Specifically, CMP-NMR technology

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incorporates magic angle spinning (MAS), a magic-angle gradient, a lock, full susceptibility

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matching, and solid-state circuitry to permit high power handling (see supporting information

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S.1 for more detail). The resulting technology permits an uncompromised analysis of liquid,

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semi-solid and solid components within intact and unaltered samples. With CMP-NMR, samples

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are deposited directly in the sample rotor and a lock solvent is added. The lock can be added

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directly to the sample or separately from the sample in an external capillary so as not to

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perturb the natural state. The goal of CMP-NMR is to permit the uncompromised study of

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structure and interactions in situ.

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METHODS Sample Preparation Xylan, alkaline lignin extract, and bovine serum albumin were purchased from Sigma

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Aldrich and were used without any modification. The lipid extract was extracted from pine

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needles with the extraction procedure noted elsewhere.22 These samples (35mg) were swollen

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with 70 µL of DMSO-d6. The soil sample used in all experiments was obtained from Hampstead

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Park in Dublin, Ireland. Hampstead Park soil is a Grey Brown Podzol with an organic carbon

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content of 6.85% ± 0.42% C, 0.59% ± 0.04% N, 4.4433% ± 0.4742% Fe, 0.0598% ± 0.0098% Mn,

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retrieved from an open public area located within Albert College Park, Dublin. 70 µL of DMSO-

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d6 was added to 35 mg of soil for the 1D 1H experiments and 2D HSQC. For the neutral soil

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sample, the same procedure was repeated using D2O. Acidic and basic soil samples were made

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by adding 65 µL of D2O to soil and then adding 5 µL of either NaOD or DCl. The pH was

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measured using a glass electrode and adjusted for the deuterium isotope effect. In acidic

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conditions, the pH equivalent was 1.8 and in basic conditions, it was 9.3.

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For solid state cross polarization (CP) experiments, each sample was run with 35mg of

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soil. The D2O and DMSO-d6 samples were prepared as described above. The dry sample was

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homogenized with solid NaCl to dilute the total mass of soil to 35mg. This approach has been

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previously developed and permits a solid sample to be “diluted” in the solid phase.23 By

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“diluting” the solids sample with NaCl (solid-state) the signal can be directly compared to the

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dilution with a liquid solvent such that the effects of the solvation on the various components in

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the sample can be easily studied and determined by difference. Throughout this study 35mg to

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70µL was chosen as the sample/solvent ratio. This represents the solvent being in excess and



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allows all potentially swell-able components to swell giving insight into the total solvent-soil

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interface. Future CMP-NMR studies using different ratios of water/soil could be performed and

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such studies could provide interesting information on the kinetics and mechanism of soil

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swelling.

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NMR Spectroscopy NMR experiments were performed on a 500 MHz Bruker Avance III Spectrometer using

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a prototype MAS 4 mm 1H-13C-2H CMP-NMR probe fitted with an actively shielded MAS

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gradient. All samples were spun using a spinning speed of 6666 Hz and all experiments were

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performed at 298K. Full details of the spectral editing methods, diffusion, T2, IT2, CP etc. are

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provided in supporting section S.2. The editing methods employed are spectroscopic based and

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do not involve changing the sample in any way. Once the samples were prepared (as described

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above), all spectral editing approaches were performed on the exact same sample.

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RESULTS AND DISCUSSION Two Dimensional (2D) Structural Characterization by CMP-NMR Before soil can be understood in its natural state it is critical to decipher the structural

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components present. This is best accomplished using a solvent to universally swell all

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components in soil. Dimethyl sulfoxide (DMSO) is a polar, aprotic solvent capable of breaking

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hydrogen bonds, and penetrating both polar and hydrophobic domains. This allows a relatively

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comprehensive swelling of soil in turn permitting structural assignment through high resolution

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“gel-state” 2D NMR experiments.24 The identification of the main structural components in soil

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is a critical precursor to better understand conformation and organization in more

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environmentally relevant aqueous systems later in the study. Figure 1 displays the two

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dimensional (2D) heteronuclear single quantum coherence (HSQC) spectra of four model

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compounds chosen to represent the main structural categories (see supporting S.3 for more

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information) that are known to contribute to SOM.11 HSQC provides connectivity information

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between directly bonded 1H and 13C units. In simple terms it can be thought of as a high

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resolution (~200,000 peak capacity)25 fingerprint of the H-C framework in a complex mixture.

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The overlay compared to an HSQC spectrum of a whole soil (Figure 1A) shows that SOM

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is reasonably well represented by a complex mixture of various biopolymeric materials that are

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abundant in plant and microbial soil inputs. Figure 1B shows two overlays against the whole

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soil, one for the biopolymer model generated using the HSQC spectra from Figure 1A and the

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other for the NMR simulation of a cross-linked humic model from Schulten and Schnitzer (see

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Figure S1), historically one of the more accepted models of humic acid.26 The goal is not to



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disprove or prove the cross-linked model but to simply demonstrate that if these “traditional”

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materials were abundant, they could be easily distinguished by HSQC NMR. Indeed it is clear

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that the spectrum of DMSO swollen organic matter (itself representing the majority of the

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SOM, see later in this article for further discussion) closely matches the spectra of the

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biopolymers. Conversely, while many peaks from the cross-linked model do match the NMR

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data, as the model contains biopolymer fragments, the majority of the peaks do not fit as a

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result of the cross-links introduced between the structural fragments.

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As such, the data indicate that in this whole soil the majority of the SOM is more

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consistent with a very complex mixture of microbial and plant biopolymers and their

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degradation products, rather than a distinct category of cross-linked humic materials. This is the

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same conclusion that was reached in earlier work which studied extracted humic materials

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from the IHSS Peat.11 In this earlier study the authors concluded in solution-state NMR

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spectroscopy that “nearly all of the NMR signals in traditional humic fractions could be assigned

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to intact and degrading biopolymers and their degradation products.” However, in this earlier

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study only the extractable humic materials were considered with the humin fraction not

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considered. In contrast, this study uses a whole grassland soil with a much lower and a more

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common carbon content of 6.8%. As the same conclusions were reached with this very

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different soil it suggests that SOM may be best thought of as complex mixture rather than a

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distinct chemical category. It is important to stress that this does not rule out the existence of

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cross-linked humic molecules at low concentrations (below NMR detection limits) or in

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different soils. These results simply indicate that in this soil, the vast majority of the SOM is

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more closely related to the logical plant and microbial inputs likely present at various states of


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oxidation and decay rather than a distinct category of cross-linked humic molecules. Future

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research should include more standard soils and in particular aged soils which could potentially

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hold high higher quantities of more traditional cross-linked humic molecules.

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Confirming the Molecular Make-up Using 13C CP-MAS NMR HSQC provides a high resolution fingerprint of soil biomolecules that allows for a

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relatively easy structural assignment. However, the drawback of HSQC is that only swollen

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domains are observed. Later in the manuscript the fraction of soil impenetrable by DMSO is

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considered in more detail. For now, the quantity of the total soil carbon that is consistent with

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the combination of the four main categories of compounds identified by HSQC in the previous

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section is explored. This is most easily investigated by employing solid-state NMR.

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In its most basic form, cross polarization magic angle spinning (CP-MAS) when applied to

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a dried soil will provide a compositional overview of all the carbon in a sample. The CP-MAS

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NMR spectra of the four biopolymers overlaid (Figure 2A) closely matches that of the soil

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confirming that the soil is consistent with a complex mixture dominated by and derived from

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carbohydrates (~30%), proteins (~30%), lignin (~30%) and lipids (~10%).

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The major difference is that the phenolic region shifts to slightly higher chemical shifts in

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the soil vs. the lignin standard. One explanation, could be that the lignin in the soil is more

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oxidized than the “fresh” lignin standard, with the additional oxygen causing nuclear

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deshielding and shifts to high ppm values. An alternate and simpler explanation could be the



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lignin in the soil and standard have a different chemical composition which gives rise to the

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slight shift. A more quantitative analysis of the component CP-MAS spectrum is provided in the

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supporting section S.5.

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Penetrability of SOM Biopolymers With a reasonable estimate of the major structural components in soil, it is possible to

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use CMP-NMR spectroscopy to extract some basic information of the solvent accessibility of the

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various soil biomolecules. Solid-state NMR provides an interesting start point in this

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endeavour. CP-MAS can be used to probe the constituents of a soil that are water accessible as

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these components will become attenuated compared to other SOM biopolymers.

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C CP-MAS is a solid-state NMR technique that transfers magnetization from proton to

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carbon via a dipolar network. In a completely dry sample, CP-MAS is very efficient as it relies on

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permanent H-C dipoles which exist in solid structures. However, upon swelling, if water

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penetrates a structure then local dynamics are introduced which modulate the H-C dipolar

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interactions in turn reducing CP-MAS efficiency. In our previous work, we have shown that for

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solution-like compounds no CP-MAS signal is observed while mobile gels are strongly

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attenuated.13

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Figure 2B shows a 13C CP-MAS overlay of a dry and hydrated whole soil, the same

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grassland soil sample used in Figure 1. The dry soil represents the compositional overview of all

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the carbon in the sample. After the addition of water, the aliphatics, carbohydrates and the


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carbonyl carbons preferentially gain molecular mobility after hydration indicating they are

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readily available at the soil-water interface. Conversely, the aromatic region (mainly from lignin)

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does not change upon swelling indicating this material is not accessible to water as is previously

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described.27 There could be three potential explanations for this. First, the lignin

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macromolecules could be mainly associated with large impenetrable plant fragments such as

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pieces of wood.28, 29 Second, they could be buried away from the surface of the soil interacting

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directly with mineral surfaces which have been shown to be layered with organic matter.28, 30

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Lastly, the components are highly hydrophobic and water cannot penetrate them.31

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It is important to note that the aliphatics, carbohydrates and carbonyl groups are not

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completely attenuated indicating a good portion of these constituents remain in the solid

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domain post-swelling. Quantification demonstrates that ~25% of the SOM is exposed at the

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water interface leaving ~75% of the material not in contact with water.

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The lower panel (Figure 2C) is similar to the one above except that DMSO was used as a

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solvent that can swell both hydrophilic and hydrophobic domains and penetrate deeper into

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the soil. Overall, ~55% of the signal has been attenuated by soil biomolecules swelling and

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becoming mobile while ~45% still remains. However it is important to stress that many gels,

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especially rigid gels, can still undergo CP-MAS, and the fraction undergoing CP-MAS after DMSO

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addition, will represent both rigid-gel and true solid structures. Therefore, the actual true

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crystalline solid and inaccessible portion of soil is likely much smaller than 45%. With DMSO as a

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solvent, the aromatic constituents are swollen while in the water hydrated soil, they were not.

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Similarly while the aliphatic components are partially swollen in water, they are more so in



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DMSO. This demonstrates that these groups are more accessible to DMSO than water and one

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reason is that water cannot penetrate these domains is due to their hydrophobic properties.

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Investigating Soil Biopolymers Based on Mobility Using 1H CMP-NMR

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Solid-state CP-MAS based approaches provide an excellent overview of changes induced

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in the total carbon with swelling. Conversely, 1H detection methods can provide more detailed

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and selective information about the critically important soil-aqueous interface.24 This

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phenomenon arises because proton signals in true solids are too broad to observe using

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conventional low-power solution-state experiments due to extensive spectral broadening

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resulting from strong 1H-1H dipoles in the solid-state. In swollen domains these interactions are

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modulated by solvent induced motion resulting in the swollen components being selectively

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detected.21 It is possible to estimate how much of the total soil organic matter is swollen by

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water based on the CP-MAS data (Figure 2B). In this case of water (neutral pH) approximately

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25% of the carbon becomes too mobile to observe by cross-polarization. In contrast about 55%

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of the carbon becomes dynamic when DMSO is used for swelling (Figure 2B and 2C). Previous

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studies have shown that gels are detected twice by both 1H MAS (specifically emphasized in 1H

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RADE experiments) and CP-MAS (specifically emphasized in T2 filtered CP).21 As such, 1H NMR

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will see everything too mobile to be detected in CP plus an additional fraction that is mobile

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enough for 1H detection but still rigid enough for CP. Hence, if the 25% of the total signal is lost

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on swelling with water from the CP-MAS experiment it is fair to say that at least 25% of the

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SOM 1H signal will be observed in the corresponding 1H experiment. Unfortunately, it is not


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possible to provide more accuracy than this based on the data at hand. However, “at least”

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approximations are provided for all spectra in Figures 3-5 such that the reader can gauge “at

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least” how much of the total organic matter is swollen and thus detected by 1H NMR under the

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varying solvent conditions and experiments reported here. Values for the % approximations for

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the 1H NMR are given with the “>” prefix to indicate the “at least” aspect.

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Figure 3 compares spectral editing approaches based on molecular self-diffusion (see

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supporting S.6 for details of the experiments). In diffusion based editing, it is the self-diffusion

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of the entire molecule (not sub-units within the molecule) that give rise to spectral

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discrimination. Figure 3A is a simple reference spectrum (no editing), contains signals from all

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components at the soil-water interface and is dominated by aliphatics and carbohydrates in an

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approximate 70:30 ratio based on 1H NMR integral. This spectrum represents >25% of the total

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organic matter (as discussed above when compared to the loss in CP on swelling with water)

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and indicates at the soil/water ratios used here (saturation) at least one in every four protons is

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in proximity to water.

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Figure 3B highlights just the molecules with free diffusion (i.e. truly dissolved) and

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contains signals from carbohydrates but not lipids. Both carbohydrates and lipids are present at

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the interface (they both appear in Figure 3A) but only carbohydrates demonstrate uninhibited

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diffusion, which suggests the lipids while swollen are associated with other soil components.

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Using iterative fitting (see supporting section S.7) it is possible to estimate the signals in the

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sub-edited spectra (Figures 3B, C, D) contribute to the total 1H. This is fitting procedure is not

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obvious from Figure 3 alone and discussed in the supporting section (S.7). This fraction



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represents approximately >7% of the total SOM. This shows that there are not many

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components in soil that are freely diffusing in comparison with semi-solid or rigid components.

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Because of continuous rain cycles, it makes sense that much of the freely diffusing species will

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be washed away.

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Figure 3C highlights the species with restricted diffusion (i.e. macromolecules, surface

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bound components, swollen polymers). Lipids are prominent confirming that they are exposed

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but not dissolved at the interface (see supporting information S.7). The exact form of the lipids

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is not clear but could involve free lipids sorbed to other soil components, and/or lipid micelles.

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This fraction again represents approximately >7% of SOM and is likely a representation of

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species that are at the soil-water interface.

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A significant contribution from carbohydrates is also apparent indicating swollen as well

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as dissolved carbohydrates also exist at the interface. Figure 3D represents biomolecules that

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are most rigid/semi-solid. This experiment termed Recovering relaxation losses Arising from

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Diffusion Editing (RADE) accounts for semi-solid components that relax too fast to be observed

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by the diffusion experiments.21 The spectrum shows a relatively similar contribution from both

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lipids and carbohydrates in the semi solid state which is consistent with their large contribution

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to the CP-MAS after swelling (Figure 2B) and likely arises, in part, from cell walls and lipid

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membranes. This fraction represents a larger portion of swollen SOM at approximately >12% of

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total SOM.

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To further probe the soil-water interface, relaxation filtered NMR can be applied. Relaxation editing differs from diffusion-based editing by providing information about local


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dynamics within a molecule rather than the diffusion of the entire molecule (see supporting S.6

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for more details). Figure 4A shows the reference spectrum of the soil (no editing) and as

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discussed above represents >25% of the total soil organic matter. Figure 4B shows the

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components with fast local dynamics which include those components with considerable

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flexibility and are oriented towards the water phase. This component accounts for

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approximately >12% of the total SOM pool, or almost half of the swollen SOM. For the

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diffusion data (Figure 3B) it was found that 7% of the organic matter was free to diffuse. This

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7% will certainly contribute to the 12% observed as flexible by relaxation editing (Figure 4b) and

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the remaining 5% must be highly flexible (i.e. seen in the T2 spectrum) but not free to diffuse.

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Such species are likely anchored to other components but oriented towards the water phase.

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Consistent with the diffusion data, the spectrum is dominated by carbohydrates.

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However, relaxation-based editing (Figure 4B) also shows some signals from aliphatics which

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have been identified as lipids (see supporting section S.8). First, it is important to note that no

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lipids were found to be freely diffusing and the diffusion data suggested these components

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were swollen but in association with other soil components. In addition, the hydrophilic end of

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the lipid is emphasized in the spectrum indicating that the COOH group of the lipid is facing the

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water interface and has the greatest degree of freedom. The (CH2)n and CH3 groups are at a

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much lower intensity indicating these sections are more rigid and orientated away from the

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water. The inverse T2 spectrum (Figure 4C) highlights components with fast relaxation (large

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molecules and semi-solids) and confirms that swollen carbohydrate and lipids with restricted

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dynamics are abundant at the soil-water interface. Aromatic groups like lignin and

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protein/peptides are not abundant in the 1H NMR indicating they are less accessible to water at



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neutral pH. The pH dependence of the biopolymers in soil will reveal more information on their

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orientation and deserves consideration (see later). The restricted molecular motion component

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represents >12% of the total organic matter and consistent with semi-solid fraction observed in

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the RADE experiment (Figure 3D). This illustrates that while components may be immobile,

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they are still be penetrated by water and have some internal mobility. This fraction of SOM is

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limited in physical movement in whole soil but is still swollen with water likely allowing it to be

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available for degradation or transformation.

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Figures 2-4 collectively illustrate the continuum that exists in biomolecules of SOM with

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respect to molecular dynamics. More than half of the SOM has the potential to interact with

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DMSO while the rest is inaccessible possibly within larger physical fragments. Water can only

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penetrate roughly a quarter of all SOM and selectively interacts with specific groups in the soil,

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namely carbohydrates and lipids. These carbohydrates are found in all states, from free in

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solution to the bound state, whereas lipids are orienting themselves where the hydrophilic end

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is towards the water interface and the hydrophobic tail is encapsulated by other soil

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components. Aromatic groups like lignin and protein/peptides are not penetrated by water

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because previous studies have shown them to be largely incorporated within microbes which

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have been shown to be removed from the water interface.32 The pH dependence of the

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biopolymers in soil will reveal more information on their orientation.

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pH and the Soil-Water Interface


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To provide more information about the soil-water interface, studies were performed

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using varying solvent conditions. Figure 5 shows the 1H soil spectrum in four different chemical

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environments: natural pH (water), acidic, alkaline, and DMSO. The soil in acidic conditions has

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the lowest signal-to-noise ratio (S/N) of all the solvents and is dominated by contributions from

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carbohydrates in an ~60:40 ratio. This spectrum represents >10% of the total SOM pool which is

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less than half of the SOM swollen with D2O only.

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This suggests that in an acidic environment, it is more difficult to penetrate the SOM

357

interface. The majority of SOM is known to be anionic,12 and at higher pH these species will

358

become charged and repel each other opening up the organic structure such that water can

359

more easily enter. However, at lower pH these components will be neutralized allowing the soil

360

to collapse becoming more difficult to penetrate. This hypothesis is supported by the spectrum

361

at neutral pH which shows a considerable increase in signal (~140%) in turn indicating that

362

components are now more easily penetrated by water. While the lipids exhibit a small increase

363

in signal, the main increase is in the carbohydrate region (representing ~75% of the total 1H

364

integral at neutral pH) indicating these materials are well swollen at neutral pH. In

365

When exposed to base, more functional groups appear compared to the acid and water

366

environments and the total signal increases 34% compared to the D2O spectrum. In particular,

367

the lipids become water exposed (77% greater than in D2O) compared to carbohydrates which

368

remain the same. The pKa of common lipids is in the range of 4.5-8 and their ionization at

369

higher pH likely increases their potential to become swollen or dissolved.33 Interestingly a

370

background from protein/peptide also increases at high pH. The most characteristic indicators



Page 20 of 44

371

are the substantial increase in the CH3 signal (from valine/alanine rich residues) along with

372

increase in the aromatic region (aromatic amino acids). Considerable NMR work has been

373

performed on protein in soil and it has been predominately shown to be from microbial

374

biomass.34, 35 Supporting section S.9 and Figure S4 considers the protein/peptide fraction in

375

more detail along with the key peptidoglycan signal (arises from microbial cell walls). In

376

summary, signals from peptidoglycan (from microbial cell walls) and microbial protein/peptides

377

are abundant at the soil water interface at high pH but less so at neutral pH. Indeed when

378

isolated microbes are analyzed at neutral pH signals from protein/peptides are clear. However,

379

the same signals are missing in the soil at neutral pH and only become visible as the pH is

380

increased (see supporting Figure S4). This suggests that the microbes may be protected by

381

other soil components (organic matter or minerals) which themselves swell at high pH

382

permitting water to contact the microbes. This is supported by work that suggests microbes are

383

largely associated with clay surfaces in whole soils.36,

384

literature that microbes may actively reside away from the aqueous-soil interface, possibly

385

associated with mineral surfaces to prevent their erosion.38, 39 The lack of accessibility to water

386

at neutral pH is consistent with such a theory.

37

It has been hypothesized in the

387

Interestingly, even at high pH the profile of the aromatic region (Figure S4b) of the soil is

388

dominated by signals from microbial protein/peptides suggesting lignin has little exposure to

389

aqueous solvents in the whole soil. This could be in part due to physical protection (for example

390

the lignin is within large pieces of plant biomass) or that its hydrophobic nature repels the

391

water making it difficult to swell. Interestingly, humic substances are generally extracted in

392

base and do contain lignin residues.40 It is likely the additional agitation (shaking) or prolonged


Page 21 of 44


393

exposure to base (possible oxidation) leads to the partial solubilisation of such components

394

during such extractions.

395

Figure 5D shows the spectrum in DMSO with a total of 120% more signal over D2O

396

(>55% of the total SOM seen in DMSO when compared to just >25% in D2O). DMSO has the

397

ability to penetrate into both hydrophilic and hydrophobic domains. In comparison to the

398

aqueous solvents used, the aliphatic portion of the spectrum is much more prominent and a

399

clear spectral envelope for protein/peptides is also apparent. Proteins in the presence of strong

400

lipid signals have been previously assigned in detail to soil microbial cells32 and in this case

401

these signals combined account for ~80% of the 1H signal indicating that on a mass basis the

402

DMSO swollen fraction is dominated by microbial biomass. Indeed DMSO is known to lyse cell

403

walls and will cause the content of microbial cells to be released.41 Interestingly this process

404

was less prominent in aqueous solvents (high pH can also lyse cells) in this study.42 The

405

implications of this are discussed more latter in “Implications to Soil Chemistry” section.

406

407

408

Piecing the puzzle together Figure 6 is an illustrative representation of the summation of the findings presented in

409

this manuscript. This figure is based entirely on the water/solvent accessibility of the various

410

soil components and further assumes the materials are layered on top of each other. In reality

411

this is likely only part of the story and many aspects such as, components buried within large

412

plant fragments, within microbial cells, associations between components and minerals are not



413

represented. Still the diagram serves a simple role in that it demonstrates, on average, the

414

proximity of the various components relative to the water interface.

415

Starting from the interface (top Figure 6), the predominant components in true solution

416

were shown to be carbohydrates. The exact identity of these components cannot be

417

determined but they likely arise in part from degradation products of parent material and soil

418

metabolism. The next regime considers (gel-like molecules) which are molecules that are

419

swollen and flexible but not free to diffuse, thus likely associate with other soil components. As

420

such these molecules represent the main soil-water interface itself. Here again carbohydrates

421

contribute indicating that within the soil there are lots of swell-able carbohydrates, logical

422

considering the range of plant materials that will be present. The polar groups of the lipids are

423

exposed to the water interface while the hydrophobic tail is less available. This suggests soil

424

lipids are orientating towards the water. The exact nature of the lipids is not clear but they

425

likely include free soil lipids as-well as cellular lipids.

426

Furthermore, this study found microbes are partially exposed when ionic associations

427

are broken under alkaline conditions and completely exposed when lysed in DMSO. At neutral

428

pH the signals from microbial protein/peptides and peptidoglycan (cell walls) are not visible at

429

the soil-water interface (Figure 3A). This suggests that microbes may be removed from the main

430

soil-water interface. This has been hypothesised in the literature and it has been suggested this

431

is to prevent their erosion.38, 39 Finally, lignin is not available to aqueous solvents and only once

432

DMSO (a strong solvent that can break H-bonds and penetrate hydrophobic domains) is

433

introduced. This suggests lignin is either physically protected (i.e. within plant cells, or buried


Page 22 of 44

Page 23 of 44


434

beneath the interface) or that it is too hydrophobic to swell and become part of the water

435

interface. Either way, lignin seems to be a likely candidate for hydrophobic contaminant

436

sequestration as a lignin moiety will offer protection from the polar water environment. The

437

authors would like to stress that the NMR data provide good insight into the orientation and

438

interaction of the SOM with respect to water in a whole soil. However, no information is

439

extracted from this current study on SOM/mineral associations or organic matter associations

440

within itself, and the key roles these surely play in aggregate soil structure. As such, Figure 6

441

should be considered as a best attempt at a diagrammatic representation consistent with the

442

results from this study rather than an accurate model of a soil. For considerations of other

443

species please consult the supporting section S.10.

444

445

446

Implications to Soil Chemistry There has been some controversy as to whether the aliphatic or aromatic components

447

of soil plays the most significant role in contaminant sorption.43-46 This study suggests that both

448

are likely important and helps support the hypothesis that both aliphatic and aromatic

449

molecules each provide hydrophobic domains that facilitate sorption of hydrophobic

450

xenobiotics.47-50 NMR evidence presented here indicates that lipid polar heads are at the soil-

451

water interface. This is consistent with earlier whole soil HR-MAS work that also demonstrated

452

lipid heads orienting towards the soil water interface.24 This suggest that just beneath these

453

polar heads there may be a hydrophobic layer of aliphatic chains that could be important for

454

sorption of hydrophobic contaminants and soil wetting properties. Non-polar contaminants will



455

likely pass quickly from the aqueous phase and could feasibly become stuck in this layer of

456

hydrophobic tails or potentially pass through (somewhat like a membrane) into a non-water

457

accessible domain.

458

Similarly, lignin residues appear impermeable to water thus once a hydrophobic

459

contaminant is introduced into the soil and finds its way to a lignin domain it may be very

460

difficult to release it. Lignin protection has been observed using other methods and has been

461

shown to bind strongly to clay surfaces which could be a cause for its differential recalcitrance

462

in soils with varying clay content and size fractions.51-55 The findings of this study are further

463

summarized in the supporting section S.11.

464

This research has shown how CMP-NMR has helped to uncover the complexity of soil by

465

revealing the organization with respect to the water interface at the molecular level using

466

samples that are in their unaltered state. Soil is a complex entity and needs to be studied in its

467

natural state. Layering, associations and general solvent accessibility are critical to unravel and

468

understand soil swelling, aggregate structure, contaminant sorption and are closely tied to

469

carbon turnover and soil fertility. Previous work has demonstrated that microbial inputs are

470

much higher than previously thought.32 Interestingly, these species are not seen at abundance

471

at the soil water interface at neutral pH but are exposed as the pH is raised. This suggests that

472

the microbial components may be protected by other soil components and once these soil

473

components are expanded at high pH water can better access the microbial components. When

474

DMSO is added the microbial components dominate the spectrum indicating that DMSO can

475

fully lyse these components. It is not clear whether DMSO is more efficient at lysing the cells

476

than high pH or if DMSO can further swell hydrophobic domains to gain better access to the


Page 24 of 44

Page 25 of 44


477

cells. Either way at neutral pH microbes are somewhat removed from the soil-water interface.

478

Microbes may actively reside away from the aqueous-soil interface, possibly associated with

479

mineral surfaces,38, 39 to prevent their erosion. Considering this, it is clear that the biology of soil

480

is equally as important as the chemistry, both of which are controlled by water accessibility and

481

the physical conformation of the materials in soil. Hence, tools such as CMP-NMR, which

482

provide the comprehensive molecular-level information in the natural state, are critically

483

needed and will likely prove essential to address many of the big questions in environmental

484

research and beyond. Future CMP-NMR studies related to soil could focus on processes for

485

example, the biodegradation of 13C enriched biopolymers such as lignin, or the binding,

486

transformation and sequestration of contaminants in-situ. Studies of living organisms should

487

provide insight into growth, adaptation and help understand contaminant stress including the

488

biochemical responses which explain toxic mode-of-action of contaminants and stressors.

489

While studies of biomass conversion, from a solid feed stage, to a liquid fuel product, could be

490

unparalleled in understanding biofuel conversion at the molecular-level. Given the importance

491

of studying intact environmental samples and that the delicate synergism between various

492

phases often determines environmental reactivity, the ability of CMP-NMR to study all

493

components in natural unaltered samples represents a key step forward for environmental

494

research and should have wide spread application in the field.



495

SUPPORTING INFORMATION Technological advances of CMP-NMR; detailed experimental

496

parameters; justification for the biopolymers chosen for the HSQC model; further explanation

497

of Figure 1; Figure S1, cross linked model; quantification of the biopolymer model; Figure S2,

498

process used to quantify biopolymer model; Table S1, chemical shift regions used for

499

integration calculations; Table S2, relative percent contribution of each model component in

500

CP-MAS; an explanation of spectral editing techniques; calculating phase contributions;

501

determining lipids to be aliphatics at the soil-water interface; Figure S3, TOCSY characterizing

502

lipids; analysis on microbe associations in soil; Figure S4, microbe analysis; components of soil

503

not mentioned; and a point form summary of findings. This information is available free of

504

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

505

ACKNOWLEDGEMENTS H.M. thanks the Natural Sciences and Engineering Research Council of

506

Canada (NSERC) for a postgraduate doctoral award and the Ontario Graduate Scholarship for

507

funding. A.J.S. thanks NSERC, (Strategic and Discovery Programs), the Canada Foundation for

508

Innovation (CFI), and the Ministry of Research and Innovation (MRI) and Krembil Foundation for

509

providing funding. A.J.S. also thanks the Government of Ontario for an Early Researcher Award.

510

B.K thanks the Irish EPA for funding through the STRIVE programme.


Page 26 of 44

Page 27 of 44


References

511

512

1. Brady, N.C. and Weil, R. The Nature and Properties of Soil. Prentice Hall: 2007;

513

2. Jenkinson, D. S.; Adams, D. E.; Wild, A.; Model Estimates of CO2 Emissions from Soil in

514

515

Response to Global Warming. Nature. 1991, 351 (6324), 304-306.

3. Kirschbaum, M. The Temperature-Dependence of Soil Organic-Matter Decomposition, and

516

the Effect of Global Warming on Soil Organic-C Storage. Soil Biol. Biochem. 1995, 27 (6),

517

753-760.

518 519

4. Lal, R.; Follett, F.; Stewart, B. A.; Kimble, J. M.; Soil carbon sequestration to mitigate climate change and advance food security. Soil Sci. 2007, 172 (12), 943-956.

520

5. Kaiser, J. Wounding earth's fragile skin. Science. 2004, 304 (5677), 1616-1618.

521

6. Baek, S.; Field, R. A.; Goldstone, M. E.; Kirk, P. W.; Lester, J. N.; Perry, R.; A Review of

522

Atmospheric Polycyclic Aromatic-Hydrocarbons - Sources, Fate and Behavior. Water Air

523

Soil Pollut. 1991, 60 (3-4), 279-300.

524 525

526

7. Heberer, T. Occurrence, fate, and removal of pharmaceutical residues in the aquatic environment: a review of recent research data. Toxicol. Lett. 2002, 131 (1-2), 5-17.

8. Kolpin, D.; Furlong, E.; Meyer, M.; Thurman, E.; Zaugg, S.; Barber, L.; Buxton, H.;

527

Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams,

528

1999-2000: A national reconnaissance. Environ. Sci. Technol. 2002, 36 (6), 1202-1211.



529 530

531 532

533 534

535 536

537 538

539

9. Okx, J.; Hordijk, L.; Stein, A.; Managing soil remediation problems. Environ. Sci. Pollut. R. 1996, 3 (4), 229-235.

10. Young, I. M.; Crawford, J. W.; Interactions and self-organization in the soil-microbe complex. Science. 2004, 304 (5677), 1634-1637.

11. Kelleher, B. P.; Simpson, A. J.; Humic substances in soils: Are they really chemically distinct? Environ. Sci. Technol. 2006, 40 (15), 4605-4611.

12. Sutton, R.; Sposito, G.; Molecular structure in soil humic substances: The new view. Environ. Sci. Technol. 2005, 39 (23), 9009-9015.

13. Derenne, S.; Nguyen Tu, T. T.; Characterizing the molecular structure of organic matter from natural environments: An analytical challenge. C. R. Geosci. 2014, 346 (3-4), 53-63.

14. Simpson, A. J.; McNally, D. J.; Simpson, M. J.; NMR spectroscopy in environmental research:

540

From molecular interactions to global processes. Prog. Nucl. Magn. Reson. Spectrosc.

541

2011, 58 (3-4), 97-175.

542

15. Conte, P.; Piccolo, A.; van Lagen, B.; Buurman, P.; de Jager, P.; Quantitative differences in

543

evaluating soil humic substances by liquid-and solid-state C-13-NMR spectroscopy.

544

Geoderma. 1997, 80 (3-4), 339-352.

545 546

16. Maas, W. E.; Bielecki, A.; Ziliox, M.; Laukien, F. H.; Cory, D. G.; Magnetic field gradients in solid state magic angle spinning NMR. J. Magn. Reson. 1999, 141 (1), 29-33.


Page 28 of 44

Page 29 of 44

547


17. Shirzadi, A.; Simpson, M. J.; Kumar, R.; Baer, A. J.; Xu, Y.; Simpson, A. J.; Molecular

548

interactions of pesticides at the soil-water interface. Environ. Sci. Technol. 2008, 42 (15),

549

5514-5520.

550

18. Semple, K. T.; Morriss, A. W. J.; Paton, G. I.; Bioavailability of hydrophobic organic

551

contaminants in soils: fundamental concepts and techniques for analysis. Eur. J. Soil Sci.

552

2003, 54 (4), 809-818.

553 554

555 556

19. Xing, B.; Pignatello, J.; Gigliotti, B.; Competitive sorption between atrazine and other organic compounds in soils and model sorbents. Environ. Sci. Technol. 1996, 30 (8), 2432-2440.

20. Alexander, M. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environ. Sci. Technol. 2000, 34 (20), 4259-4265.

557

21. Courtier-Murias, D.; Farooq, H.; Masoom, H.; Botana, A.; Soong, R.; Longstaffe, J. G.;

558

Simpson, M. J.; Maas, W. E.; Fey, M.; Andrew, B.; Struppe, J.; Hutchins, H.;

559

Krishnamurthy, S.; Kumar, R.; Monette, M.; Stronks, H. J.; Hume, A.; Simpson, A. J.;

560

Comprehensive multiphase NMR spectroscopy: Basic experimental approaches to

561

differentiate phases in heterogeneous samples. J. Magn. Reson. 2012, 217 61-76.

562

22. Kogel-Knabner, I.; Deleeuw, J.; Tegelaar, E.; Hatcher, P.; Kerp, H.; A Lignin-Like Polymer in

563

the Cuticle of Spruce Needles - Implications for the Humification of Spruce Litter. Org.

564

Geochem. 1994, 21 (12), 1219-1228.



565

23. Masoom, H.; Courtier-Murias, D.; Farooq, H.; Soong, R.; Simpson, M. J.; Maas, W.; Kumar,

566

R.; Monette, M.; Stronks, H.; Simpson, A. J.; Rapid estimation of nuclear magnetic

567

resonance experiment time in low-concentration environmental samples. Environ.

568

Toxicol. Chem. 2013, 32 (1), 129-136.

569

24. Simpson, A. J.; Kingery, W. L.; Shaw, D. R.; Spraul, M.; Humpfer, E.; Dvortsak, P.; The

570

application of H-1 HR-MAS NMR spectroscopy for the study of structures and

571

associations of organic components at the solid - Aqueous interface of a whole soil.

572

Environ. Sci. Technol. 2001, 35 (16), 3321-3325.

573

25. Hertkorn, N.; Ruecker, C.; Meringer, M.; Gugisch, R.; Frommberger, M.; Perdue, E. M.; Witt,

574

M.; Schmitt-Kopplin, P.; High-precision frequency measurements: indispensable tools at

575

the core of the molecular-level analysis of complex systems. Anal. Bioanal. Chem. 2007,

576

389 (5), 1311-1327.

577 578

579

26. Schulten, H. R.; Schnitzer, M.; A State-Of-The-Art Structural Concept for Humic Substances. Naturwissenschaften. 1993, 80 (1), 29-30.

27. Conte, P.; Berns, A. E.; Dynamics of cross polarization in solid state nuclear magnetic

580

resonance experiments of amorphous and heterogeneous natural organic substances.

581

2008, 24 (9), 1183-1188.

582 583

28. Baldock, J. A.; Skjemstad, J. O.; Role of the soil matrix and minerals in protecting natural organic materials against biological attack. Org. Geochem. 2000, 31 (7-8), 697-710.


Page 30 of 44

Page 31 of 44


584

29. Baldock, J. A.; Oades, J. M.; Nelson, P. N.; Skene, T. M.; Golchir, A.; Clarke, P.; Assessing the

585

extent of decomposition of natural organic materials using solid-state 13C NMR

586

spectroscopy. Aust. J. Soil Res. 1997, 35 (5), 1061-1083.

587

30. Kleber, M.; Sollins, P.; Sutton, R.; A conceptual model of organo-mineral interactions in soils:

588

Self-assembly of organic molecular fragments into zonal structures on mineral surfaces.

589

Biogeochemistry. 2007, 85 (1), 9-24.

590

31. Song, X. Y.; Spaccini, R.; Pan, G.; Piccolo, A.; Stabilization by hydrophobic protection as a

591

molecular mechanism for organic carbon sequestration in maize-amended rice paddy

592

soils. Sci. Total Environ. 2013, 458-460 319-330.

593

32. Simpson, A. J.; Simpson, M. J.; Smith, E.; Kelleher, B. P.; Microbially derived inputs to soil

594

organic matter: Are current estimates too low? Environ. Sci. Technol. 2007, 41 (23),

595

8070-8076.

596 597

33. Kanicky, J. R.; Shah, D. O.; Effect of degree, type, and position of unsaturation on the pKa of long-chain fatty acids. J. Colloid Interface Sci. 2002, 256 (1), 201-207.

598

34. Simpson, A. J.; Song, G.; Smith, E.; Lam, B.; Novotny, E. H.; Hayes, M. H. B.; Unraveling the

599

structural components of soil humin by use of solution-state nuclear magnetic

600

resonance spectroscopy. Environ. Sci. Technol. 2007, 41 (3), 876-883.

601 602

35. Schleife.Kh; Kandler, O.; Peptidoglycan Types of Bacterial Cell-Walls and their Taxonomic Implications. Bacteriol. Rev. 1972, 36 (4), 407-477.



603

36. Courtier-Murias, D.; Simpson, A. J.; Marzadori, C.; Baldoni, G.; Ciavatta, C.; Fernandez, J. M.;

604

Lopez-de-Sa, E. G.; Plaza, C.; Unraveling the long-term stabilization mechanisms of

605

organic materials in soils by physical fractionation and NMR spectroscopy. Agric.

606

Ecosyst. Environ. 2013, 171 9-18.

607

37. Plaza, C.; Courtier-Murias, D.; Fernandez, J. M.; Polo, A.; Simpson, A. J.; Physical, chemical,

608

and biochemical mechanisms of soil organic matter stabilization under conservation

609

tillage systems: A central role for microbes and microbial by-products in C

610

sequestration. Soil. Biol. Biochem. 2013, 57 124-134.

611 612

613

38. Kim, J.; Dong, H.; Seabaugh, J.; Newell, S.; Eberl, D.; Role of microbes in the smectite-to-illite reaction. Science. 2004, 303 (5659), 830-832.

39. Lower, S.; Hochella, M.; Beveridge, T.; Bacterial recognition of mineral surfaces: Nanoscale

614

interactions between Shewanella and alpha-FeOOH. Science. 2001, 292 (5520), 1360-

615

1363.

616

40. Stevenson, F.J. Humus chemistry : genesis, composition, reactions. Wiley: New York, 1994;

617

41. David, N. A. The pharmacology of dimethyl sulfoxide. Annu. Rev. Pharmacol. 1972, 12 353-

618

619 620

374.

42. Brown, R. B.; Audet, J.; Current techniques for single-cell lysis. J. R. Soc. Interface. 2008, 5 (SUPPL.2), S131-S138.


Page 32 of 44

Page 33 of 44


621

43. Sun, K.; Ran, Y.; Yang, Y.; Xing, B.; Sorption of phenanthrene by nonhydrolyzable organic

622

matter from different size sediments. Environ. Sci. Technol. 2008, 42 (6), 1961-1966.

623 624

625

44. Chefetz, B. Sorption of phenanthrene and atrazine by plant cuticular fractions. Environ. Toxicol. Chem. 2003, 22 (10), 2492-2498.

45. Perminova, I. V.; Grechishcheva, N. Y.; Petrosyan, V. S.; Relationships between structure and

626

binding affinity of humic substances for polycyclic aromatic hydrocarbons: Relevance of

627

molecular descriptors. Environ. Sci. Technol. 1999, 33 (21), 3781-3787.

628

46. Johnson, M. D.; Huang, W. H.; Weber, W. J.; A distributed reactivity model for sorption by

629

soils and sediments. 13. Simulated diagenesis of natural sediment organic matter and its

630

impact on sorption/desorption equilibria. Environ. Sci. Technol. 2001, 35 (8), 1680-1687.

631

47. Clemente, J. S.; Simpson, A. J.; Simpson, M. J.; Association of specific organic matter

632

compounds in size fractions of soils under different environmental controls. Org.

633

Geochem. 2011, 42 (10), 1169-1180.

634

48. Almendros, G.; Guadalix, M. E.; Gonzalez-Vila, F. J.; Martin, F.; Distribution of structural

635

units in humic substances as revealed by multi-step selective degradations and C-13-

636

NMR of successive residues. Soil Biol. Biochem. 1998, 30 (6), 755-765.

637 638

49. Almendros, G.; Guadalix, M. E.; GonzalezVila, F. J.; Martin, F.; Preservation of aliphatic macromolecules in soil humins. Org. Geochem. 1996, 24 (6-7), 651-659.



639

50. Poirier, N.; Derenne, S.; Rouzaud, J. N.; Largeau, C.; Mariotti, A.; Balesdent, J.; Maquet, J.;

640

Chemical structure and sources of the macromolecular, resistant, organic fraction

641

isolated from a forest soil (Lacadee, south-west France). Org. Geochem. 2000, 31 (9),

642

813-827.

643

51. Feng, X. J.; Simpson, A. J.; Simpson, M. J.; Chemical and mineralogical controls on humic acid

644

sorption to clay mineral surfaces. Org. Geochem. 2005, 36 (11), 1553-1566.

645

52. Clemente, J. S.; Simpson, M. J.; Physical protection of lignin by organic matter and clay

646

647 648

649 650

651 652

minerals from chemical oxidation. Org. Geochem. 2013, 58 1-12.

53. Emery, E.; Junk, T.; Ferrell, R.; De Hon, R.; Butler, L.; Solid-state H-2 MAS NMR studies of TNT absorption in soil and clays. Environ. Sci. Technol. 2001, 35 (14), 2973-2978.

54. Heim, A.; Schmidt, M. W. I.; Lignin turnover in arable soil and grassland analysed with two different labelling approaches. Eur. J. Soil Sci. 2007, 58 (3), 599-608.

55. Heim, A.; Schmidt, M. W. I.; Lignin is preserved in the fine silt fraction of an arable Luvisol. Org. Geochem. 2007, 38 (12), 2001-2011.

653


Page 34 of 44

Page 35 of 44

654


FIGURE 1

655 656

Figure 1: A) The four panels on the left show 2D HSQC spectra of the four model compounds

657

used to characterize SOM. The numbered boxes indicate regions where cross peaks of

658

important chemical bonds arise that are present in soils. The red box in the biopolymer overlay

659

highlights lignin syringyl units which are in higher abundance in the model lignin compared to

660

the grass land soil used since the model lignin is derived from woody plants. Readers should

661

understand that the goal is not to assign these exact biopolymers in the soil, but simply use

662

these biopolymers to demonstrate the spectral regions in which general classes of

663

carbohydrates, lignin, lipids and protein resonate (see supplementary information (S.3. and S.4)



664

for further discussion). Once these four spectra are overlaid, each box is filled and the spectrum

665

closely matches that of whole soil. This indicates that soil has strong contributions from

666

carbohydrates, lipids/aliphatics, protein/peptides, and lignin. B) Whole soil overlays with an

667

NMR simulation of “traditional” cross-linked humic acid from Shulten and Shnitzer and the

668

biopolymer model derived from (A). The biopolymer model closely matches that of whole soil

669

illustrating that the majority of SOM is consistent with a range of biopolymeric inputs and their

670

related degradation products rather than a distinct chemical category of cross-linked humic

671

materials.


Page 36 of 44

Page 37 of 44


672

FIGURE 2

673

Figure 2: (A) 13C CP-MAS comparison of the

674

dry whole soil sample (brown) compared to

675

the weighted summation CP-MAS spectrum of

676

the individual biopolymers used in Figure 1

677

(black). The similarities between the two

678

spectra suggest a large proportion of the soil

679

spectral envelope is consistent with, or

680

derived from, these biopolymers. (B)

681

Comparison of the dry whole soil sample

682

compared to the same sample with water

683

added and (C) DMSO instead of water used to

684

swell the soil. Carbohydrates, aliphatics and

685

carbonyl carbons are the groups that have

686

become attenuated, hydrated, and have

687

gained mobility. DMSO impacts all regions

688

demonstrating its non-discriminate nature

689

compared to water.



690

FIGURE 3

691 692

Figure 3: 1H spectra of the wet soil sample edited based on diffusion properties. (A) A reference

693

spectrum illustrating all components at the water interface. (B) Inverse diffusion editing

694

represents compounds that have unrestricted diffusivity and is dominated by carbohydrates.

695

(C) Molecules that have restricted diffusion due to macromolecular size and/or sorption.

696

Aliphatics have a large contribution likely being adsorbed at the surface of a soil colloid. (D)

697

Recovering relaxation arising from diffusion editing experiment highlights fast relaxing semi-


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698

solid/rigid compounds and have strong contributions from both carbohydrates and aliphatics.

699

The red brackets indicate an artifact from incomplete water suppression. Relative abundance

700

refers to the total soil organic matter represented in the spectrum. This is calculated as

701

described in the supporting section S.7 and should be interpreted as an “at least” value (see

702

main text for further explanation).



703

FIGURE 4

704

705

Figure 4: 1H spectra of the wet soil sample that show biomolecules with varying degrees of

706

intra-molecular motion. (A) Reference spectrum showing all components available at the water

707

interface. (B) Illustrates compounds that have dynamic molecular motion (T2 filtered), such as

708

dissolved molecules or flexible domains. (C) Shows components with restricted mobility

709

(inverse T2 filtered, A minus B), such as macromolecules swollen biopolymers. The red brackets

710

indicate an artifact from incomplete water suppression. *Acetic acid also contributes to this

711

peak. Relative abundance refers to the total soil organic matter represented in the spectrum.


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712

This is calculated as described in the supporting section S.7 and should be interpreted as an “at

713

least” value (see main text for further explanation).

714



715

FIGURE 5

716 717

Figure 5: 1H spectra representing different functionalities that arise in whole soil after being

718

exposed to different solvent conditions. (A) Under acidic conditions the peak intensity and the

719

carbohydrate contribution is lower relative to neutral pH conditions (B). When exposed to

720

alkaline conditions (C), the intensity increases namely in the aliphatic region and the aromatics


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721

are discernible from the noise. After the addition of DMSO-d6 (D), H-bonds and hydrophobic

722

interactions are broken. The baseline of the spectrum closely resembles that of protein and

723

could be a large contribution from microbes after being lysed by DMSO. Relative abundance

724

refers to the total soil organic matter represented in the spectrum. This is calculated as

725

described in the supporting section S.7 and should be interpreted as an “at least” value (see

726

main text for further explanation).



727

FIGURE 6

728 729 730

Figure 6: A diagrammatic representation moving from close proximity to the water (top) to

731

furthest removed from the water (bottom). This figure is not a model of soil and serves a simple

732

role in that it demonstrates, on average, the proximity of the various components relative to

733

the water interface. Free in solution are carbohydrate (green) species while at the soil-water

734

interface, carbohydrates and lipids (yellow) where the polar groups are faced towards the

735

water. Not accessible to water are lignin (purple) and microbes (white) as well as other

736

carbohydrate and aliphatic molecules.


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Soil Organic Matter in Its Native State: Unravelling ... - ACS Publications

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