APPLICATIONS OF COMPREHENSIVE MULTIPHASE NMR TO

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APPLICATIONS OF COMPREHENSIVE MULTIPHASE NMR TO CHARACTERIZE ORGANIC MATTER IN CLAY AND ATMOSPHERIC PARTICULATES Bing Zhou, Yulian Zhao, and Qunwei Dai ACS Earth Space Chem., Just Accepted Manuscript • DOI: 10.1021/ acsearthspacechem.9b00148 • Publication Date (Web): 19 Aug 2019 Downloaded from pubs.acs.org on August 25, 2019

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ACS Earth and Space Chemistry

APPLICATIONS OF COMPREHENSIVE MULTIPHASE NMR TO CHARACTERIZE ORGANIC MATTER IN CLAY AND ATMOSPHERIC PARTICULATES

Bing Zhou1*, Yunlian Zhao2, Qunwei Dai2* 1. School of Material Sciences and Technologies, Tongji University, Shanghai 201804, China 2. School of Environment and Resource, Southwest University of Sciences and Technology, Mianyang 621010, China

*: corresponding authors [email protected] Qunwei Dai: [email protected]

Key Words: montmorillonite, CMP NMR, organic matter, microorganisms, atmospheric particulates, dynamics and surficial interactions, molecular and atomic scales

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ABSTRACT The molecular composition of organic matter (OM) and its interaction with minerals play important roles in regulating the fate of carbon and pollutants in soil and in the atmosphere. Clay minerals such as montmorillonite processed with bacteria can imitate the microbial interactions occurring in more complex natural environmental media such as soil and atmospheric particulates. Here, we characterize OMs in multiple phases for bacteria-inoculated montmorillonite clay and atmospheric particulates collected in China. In addition to the traditional 13C Cross-Polarization Magic Angle Spinning Nuclear Magnetic Resonance (CPMAS NMR) experiments, this study, for the first time, applies both one and two-dimensional Comprehensive Multi-Phase (CMP) NMR to these critical environmental issues. The CMP NMR experimental spectra show that the main OM components in these samples are aliphatics, carbohydrates and aromatics. These CMP NMR spectra also reveal the biochemical interactions between microbes and clay minerals by identifying the metabolites. CMP NMR can further investigate the states, interfacial/surficial interactions and dynamics at the molecular/atomic levels, since diffusion/relaxation (T2)-edited experiments can distinguish OM with differing size and mobility. Therefore, the mechanism at the atomic/molecular level for environmental biogeochemical interactions can be efficiently probed by CMP NMR experiments. This paper concludes that CMP NMR is a powerful and innovative tool for crucial environmental studies at the molecular/atomic scale, providing not only the composition, phase and structural identifications, but also the interfacial/surficial interaction dynamics involved in environmental geochemical processes.


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1. INTRODUCTION Climate change and environmental pollution from emerging contaminants are of increasing concern to scientists and the general public. Understanding the composition of organic matter (OM) and its interaction with minerals at a molecular and atomic level is essential to understand the chemical stabilization of organic carbon and sequestration of environmental pollutants.1 For example, soil OM bound to minerals can be protected from microbial degradation and, therefore, less likely to be transformed into greenhouse gases. Similarly, pollutants bound to the OM-mineral complexes usually have lower mobility and bioavailability. Therefore, it is important to understand these environmental interactions at an atomic level.

2,3

Most research has focused on the

concentration, chemical composition, physical property and predominant source, 4-10 more studies are in need to investigate the interactions between OM and minerals. Complex environmental issues, concerned with chemical structure and interactions of soils and atmospheric particulates, can only be answered through understanding the molecular-level framework that underlies the problems. 1,11 The key advantage of nuclear magnetic resonance (NMR) lies in providing direct measurements, because it can directly detect the molecular/atomic scale phenomena and can provide such information, even for the complex and multiphase samples which are ubiquitous in environmental research1,11. The potential impact of NMR on environmental research is growing due to the steadily increasing sensitivity of NMR experiments 1,11,12. Traditionally, NMR has developed in two separate fields, i.e., one dealing with solids and the other with liquids, however, neither is ideal for studying complex and multiphase environmental



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samples. 13 Technological advances, such as High Resolution Magic Angle Spinning NMR (HRMAS NMR) allow non-rigid solid materials (gels) and highly viscous and anisotropic liquids to be studied in a range of applications. Silva et al.

13

pointed out one major drawback of the

conventional HR-MAS NMR probes is that it can only handle low power Radio Frequency (RF) pulses, but cannot generate the intense RF fields required for high power decoupling or CrossPolarization (CP) NMR to study true solid phases in samples. On the other hand, in order to fully understand the structures, phases, dynamics, environmental interactions and processes at a molecular level, it is critical to study environmental samples in their natural and relatively intact state, containing all the different phases of solution, gel and solid. Recently a novel approach, namely Comprehensive Multiphase NMR spectroscopy, CMP-NMR, has been introduced to study the different phases coexisting in complex samples.

1,13,14

This innovative methodology uses a

probe head constructed with high-power circuitry, magic angle spinning (MAS), full magnetic susceptibility matching, a lock channel, and pulse field gradients. Therefore, this probe incorporates crucial aspects from low power solution-state NMR probes with the hardware common to solid-state probes. Thus, high speed MAS and high power circuitry for true solids are combined with setups for solutions, such as a lock to provide stability over time, magnetic susceptibility matching to provide excellent line shape, pulse field gradients to permit coherence selection and diffusion measurements, as well as advanced water suppression for solution.

1,13,14

Therefore, the CMP probe allows the study of all phases within sample in their native state without any physical separation. The components in gel and solution phases can be relatively easily distinguished by using T2-relaxation Editing (TE) and Diffusion Editing (DE), while CP can investigate the solid phases via strong dipolar interaction. 1,11 This new approach has enabled the study of chemical compositions and structures as well as the interactions in complex multiphase


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samples. In conclusion, CMP NMR, which integrates solution, gel, and solid state NMR, provides a comprehensive overview of all phases in environmental samples in their natural state. 1 This study used CMP-NMR to study the components of interest in all phases to understand the origin, dynamics, interactions, and fate of organic components in samples of clay and atmospheric particulates. Information about environmental reactivity as well as the effects of these organics and interactions can be found. Clays are the main components of soil and atmospheric particulates and determine the main properties of these environmental media. Pure montmorillonite was chosen as a model mineral due to its strong adsorption ability, large specific surface area and swelling capacity. To investigate the biochemical interactions involved with clays, we cultured bacteria in a montmorillonite suspension in the laboratory to mimic the biological interactions with particulates in the atmosphere and in soil. These montmorillonite studies using CMP NMR can provide the crucial information for soil and smog environmental issues. CMP NMR is shown to be an innovative and powerful tool with great potential for environmental geochemical studies.

2. MATERIALS AND EXPERIMENTAL PROCEDURES 2.1. Collection of atmospheric particulate samples. We collected dust fall during the period of April-May 2016, when sandstorms occurred frequently and catkins provided fibrous materials in air dust. Atmospheric particulate samples were collected along the boundary of sandstorm areas in China, including Zhangjiakou city (Sample #2, Hebei Province), Tianshui city (Sample #3, Gansu Province), Xining city (Sample #4, Qinghai Province), Tuoketuo city (Sample #5, Inner Mongolian Autonomous Region), and also far away from the sandstorm area in Mianyang city (Sample #6, Sichuan Province). These samples



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were collected using brushes and water-retaining devices for keeping them dry. A 400-mesh nylon sieve was used to exclude large particles. The fine particulates were then sealed in dry and cool containers. The total organic carbon (TOC) in each sample was determined by a standard potassium dichromate oxidation method (Soil-determination of Organic Carbon-potassium Dichromate Oxidation Spectrophotometric Method stipulated by China’s State Environmental Protection Administration (CEPA) in 2011). 2.2. Sample preparation for bacteria-inoculated montmorillonite. Montmorillonite was collected from the Altay Area of Xinjiang Uygur Autonomous Region in China. The sample was purified by repeated suspension with Milli-Q water and centrifugation,

15,16

then ground and sieved using 200-mesh to prepare the powder sample with

grain sizes < 75 μm for XRD and XRF analysis. Bacillus pumilus (B. pumilus, GenBank Accessing Number: EF512718), a normal inhabitant in meadow soil, was isolated from a lawn soil in the Sichuan Basin of China. It was identified by its morphology and 16s rRNA gene analysis.

17,18

The bacterial cell is 0.6~0.7

μm×2.0~3.0 μm. The bacterial culture was composed of 6.0 g/L glucose, 3.0 g/L beef extract, 10.0 g/L peptone and 5.0 g/L NaCl, and maintained on agar slants and stored at 4.0°C. The pH of the culture solution was adjusted to 7.0-7.2 with hydrochloric acid or sodium hydroxide. B. pumilus was incubated in 250 mL Erlenmeyer flasks containing 100 mL of the culture medium at 35.0 °C and shaken at 150 rpm/min. After cultivation for 0, 1, 2 or 3 d, the 1.6 g/L montmorillonite suspension with or without dialysis bag was added to the systems. Extracellular polymeric substances (EPS) were then isolated from B. pumilus by following the procedures described by Omoike & Chorover 19.


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The sugar culture medium and montmorillonite were sterilized at 121°C for 20 min by high-pressure steam before NMR sample preparation. There were two kinds of sample preparation, i.e., with and without EPS. Preparation for samples without EPS: 1.6 g/L montmorillonite stock suspension was enclosed in a dialysis bag (36 MM, MW of 8000-14000). After interacting with B. pumilus, the suspension was centrifuged at 10,000 rpm for 10 min, washed 3 times using ultrapure water (18.0 MΩ·cm) to remove the bacteria cells and EPS, then dried in an oven at 65°C for 1 day and finally ground to a powder. Preparation for samples with EPS: 1.6 g/L montmorillonite suspension was added to the 100 mL of culture medium in 250 mL Erlenmeyer flasks. Cells were removed from the culture solution by centrifugation at 10,000 rpm for 10 min, and the supernatant solution was centrifuged at a higher force to remove residual entrained cells. EPS was then precipitated from the supernatant solution by adding cold reagent-grade ethanol, and the precipitate was separated from the ethanol suspension by centrifugation for 30 min. Three pellet obtained after centrifugation was washed 3 times using ultrapure water (18.0 MΩ.cm), dried in an oven at 65°C for 1 d and finally ground to a powder. As a result of this preparation, Clay #19 is a blank contrast sample of Nano-SiO2 without interaction with any microorganism, Clay #20 is montmorillonite which interacted only with the culture medium without bacteria for 1 d; Clay #21 is montmorillonite which interacted with B. pumilus by dialysis bag for 1 d without EPS, and Clay #22 is from the interaction between montmorillonite and EPS of B. pumilus for 1 d, while Clay #23 is from the interaction between montmorillonite (1.6 g/L) and EPS of B. pumilus for 3 d.



2.3. XRD and XRF experiments. Powder XRD data were collected at room temperature with a computer-controlled XRD (X’pertPRO) at The Analysis and Testing Center of Southwest University of Science and Technology, using CuKa radiation with a voltage of 40 kV and current of 40 mA, and a continuously scanning speed with DS 1/2°, SS 0.04 rad, AAS 5.5 mm. The chemical composition of montmorillonite was measured by XRF (Axios) at The Analysis and Testing Center of Southwest University of Science and Technology, while atmospheric particulate samples were analyzed by XRF (PW2404) at The Geological Analysis and Testing Center of Nuclear Industry in Beijing. 2.4. Conventional 13C CPMAS NMR experiment. Conventional 1H-13C CPMAS NMR spectra for clay samples processed with bacteria were recorded with a 4-mm H-X MAS probe at room temperature on a Bruker Avance III NMR spectrometer at 1H 600 MHz, using a MAS frequency of 13,000 Hz, a recycle delay of 1 s, a rampCP contact time of 1 ms, and between 2112 and 225,280 scans. The free induction decay (FID) signal was digitized, processed with zero-filling factor of 2, and multiplied by an exponential function corresponding to 50 Hz line broadening in the final transformed spectrum. Spectra were calibrated using the carboxyl signal of glycine as an external standard (176.03 ppm). 2.5. CMP NMR experiments. All CMP NMR spectra were acquired on a Bruker Avance III NMR spectrometer at 1H 500.28 MHz, and at 298 K in the University of Toronto, using a prototype 2.5 mm QXI (1H-2H19F-13C)

CMP-NMR probe fitted with full power solid-state capability, an actively shielded magic

angle gradient coil, and susceptibility matched stator to achieve high resolution line shapes. All


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samples were spun at a rate of 6666 Hz. Before the CMP NMR experiments, around 32 mg of dried sample was respectively dissolved in 0.8 mL of both D2O and DSMO-d6, then transferred into 2.5-mm NMR tubes (a zirconium rotor) and sealed using a Kel-F insert and a Kel-F rotor cap. Prior to the NMR analysis including the CPMAS NMR experiments, the samples were locked and shimmed using the D2O/H2O NMR signal from the lower compartment. All the data were processed in the software TopSpinTM version 2. 1H-13C

CPMAS using the CMP probe was performed with a 80-100% ramp, a contact time

of 2 ms, and SPINAL-64 1H decoupling with 70 kHz of RF strength during the acquisition. For the variable contact time CPMAS experiments, 80-100% ramp with contact time ranging from 0.5 to 4.5 ms was used. Other acquisition parameters were set as 24576 scans, and a time domain (number of raw data points) of 1024. 1D solution-state 1H CMP NMR spectra of dissolved OMs were acquired using ZGPR pulse sequence, 512 scans, a 2 s delay between pulses, a sweep width of 20 ppm, 8192 time-domain points. Presaturation Utilizing Relaxation Gradients and Echoes (PURGE) was used for water suppression. The DE 1H CMP NMR spectra were collected with a longitudinal encode-decode bipolar pulse (BPPLED) sequence using a 1.25 ms at 33.3 G cm-1 sine-shaped gradient pulse, a diffusion time of 60 ms, 4096 time-domain points and 1024 scans. To create inversion diffusion editing (iDE) spectra, a second dataset was collected with all delays set identically but with the diffusion gradient set to 0 Gauss/cm (‘‘zero-gradient”). The iDE spectrum was then obtained by subtracting the DE (restricted components) from the ‘‘zero-gradient” (the whole components) to give components with unrestricted diffusion. The 1H CMP NMR spectra were processed with a zero-filling factor of 2 and an exponential function corresponding to 1 Hz line broadening in the transformed spectrum, and then calibrated using the solvent residual peak at 2.5 ppm.



For TE experiments using the CMP probe, a pulse sequence of CPMG (T2f) was applied with an echo time of 1.2 ms and the pulse train repeated 4 or 100 times, yielding a total pulse train length of 9.6 and 240 ms, respectively. The acquired T2f data were processed by a zero-filling factor of 2, and through multiplication with an exponential decay corresponding to 0.3 Hz of line broadening. The 2D 1H-13C Heteronuclear Single Quantum Coherence (HSQC) CMP NMR spectra were obtained in phase-sensitive mode using the Echo/Antiecho-TPPI gradient selection, the pulse sequence of HSQCETGPSP-SUPER and a 1H-13C J-coupling value of 145 Hz. 512 scans, 1024 data points and a recycle delay of 0.5 s, were used for each of the 256 increments in the F1 dimension. The 2D Total Correlation Spectroscopy (TOCSY) spectra were acquired in the phasesensitive mode using the pulse sequence of ATOCSYGPPR with a mixing time of 80 ms. Scans of 2048 were collected for each of the 196 increments in the F1 dimension, while a total of 1024 data points were collected in F2 dimension with a relaxation delay of 2 s was employed. F2 planes of both TOCSY and HSQC spectra were multiplied by an exponential function corresponding to a 15 Hz line broadening, while the F1 dimensions were multiplied by an exponential function corresponding to a 0.3 Hz line broadening, and processed with a zero-filling factor of 2 and using sine-squared functions with a π/2 phase shift.

3. RESULTS AND DISCUSSION 3.1. Chemical and mineral composition of montmorillonite and atmospheric particulates. The mineral phases of the purified clay sample were determined by XRD to be composed mainly of Ca-montmorillonite with d001 of 1.486 nm and minor quartz. The chemical compositions


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of Ca-montmorillonite, analyzed by XRF, is SiO2 59.65%, Al2O3 18.06%, Fe2O3 4.22%, MgO 2.54%, CaO 1.41%, Na2O 2.92%, K2O 1.77%, MnO 0.03%, TiO2 0.39%, P2O5 0.04%, SrO 0.04% with a loss on ignition of 8.90%. The cation exchange capacity of the montmorillonite is 76.50 mmol/g. The main mineral composition of atmospheric particulate samples, detected by XRD, are clays (such as muscovite and clinochlore), quartz, calcite, dolomite and trace amount of gypsum, with a TOC between 1 and 2.0%. 3.2. Identification and characterization of OM in the bacteria-processed clay samples via 1H CMP (1D and 2D) NMR spectroscopy. Combined with NMR parameters such as spectrometer frequency, sweep width and line shape, NMR chemical shift assignments were based upon previously published data

1, 20-24

and

further confirmed by 2D CMP NMR experiments of Wheeler et al. 14. (1) 1D CMP 1H NMR spectra for clay samples A series of 1D 1H CMP NMR spectra for clay samples were acquired to investigate the OM profiles. These 1D 1H NMR spectra contain resonances spanning an entire 1H chemical shift range ~10 ppm, indicating that all the differing OM components, such as aliphatic, hydrocarbons and aromatics, occur in these samples (Fig. 1). The 1D 1H NMR spectrum for Clay #23 is significantly different from other clay samples in terms of the number and resolution of the spectral peaks, especially the peak around 3.8 ppm in the spectrum using D2O solvent (Fig. 1), which is probably from methoxyl (lignin).

1

These

differences between Clay #23 and other clay samples may be caused by its longer interaction with bacteria (3 days), producing more metabolites and much higher TOC. The longer T1 times



possessed by the smaller and so more mobile metabolites may also contribute to this higher 1H NMR spectral resolution (Simpson et al.1).

Figure 1 CMP 1D 1H NMR spectral comparisons between solvents of D2O (red) and DSMO-d6 (blue) for Clay #23

Different solvents can also significantly affect 1D 1H NMR resonances from each organic category. The 1H NMR peaks of Clay #23 using D2O as the solvent are systematically shifted to left compared with its counterparts using DSMO-d6 (Fig. 1). These spectral differences caused by using different solvents (DSMO-d6 vs D2O) reflect the polar properties of dissolved OM components, since carbohydrate components are more soluble (more hydrophilic or polar) in D2O. Simpson et al.

12

also showed such 1H NMR spectral differences due to different solvents,


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especially the polar hydrophilic constituents (such as aliphatic acids) dissolved in D2O vs their hydrophobic counterparts in DSMO-d6. As a result, the resonance at 3.8 ppm from methoxyl only occurs with D2O but not DSMO-d6 (Fig. 1), and the region for units adjacent to an oxygen, esters, ethers and alcohols in DSMO-d6 is in contrast to the predominant carbohydrates in D2O (Fig. 1). Therefore, the use of both D2O and DSMO-d6 solvents give a fuller picture of OM in the samples. (2) 2D CMP NMR spectra for clay samples 2D 1H-13C HSQC NMR can provide connectivity information between protons and carbons, while the second dimension can also provide additional spectral dispersion. It can be used to give a more comprehensive overview of the organic profile, and can also provide a detailed ‘fingerprint’ of a wide range of metabolites in samples. 14 2D 1H TOCSY spectra can highlight the major and longer range 1H-1H couplings, which cause further spectral dispersion and are useful in assigning NMR signals, especially for metabolites. HSQC and TOCSY spectra for Clay #23 display all the microbial signatures (Figs. 2 & 3). The main cross peaks in the 1H-13C HSQC spectra obtained by CMP NMR (Fig. 2) are assigned by a combination of 1D NMR spectra with the schemes of Simpson et al., 1 Plaza et al.

25

and

Pautler et al. 26. Consequently, the region centering around (7.2, 130) at the left-lower corner in the 1H-13C HSQC spectrum (Fig. 2) is from phenylalanine (Phe, peptide); the peak at (6.7, 116) mainly results from tyrosine (Tyr, aromatic protons adjacent to an Ar-OR functionality in lignin); the peaks centering around (4.3, 52) and (3.8, 43) are caused by α-CH in peptides/proteins; other weak CH signals from carbohydrates contribute to the regions of (3~5, 70~85); lastly, the large aliphatic linkage groups give rise to the region at the right-upper corner in the spectrum, including signals from various lipids and side-chain protons in peptides. Additionally, from left to right along the 1H chemical shift axis, large aliphatic group can be further assigned into sub-groups: R-OCO-



CH2-R methylene unit adjacent to the carbonyl in lipids (including lipoproteins and cutins); CH methylene units in an aliphatic chains to an β-acid or ester; CH methylene units in an aliphatic chains to an γ-acid or ester; (CH2)n methylene in aliphatic chains; CH3 methylene units mainly in peptides (partly from terminal CH3 in lipids). There is a lack of resonances from units such as anomeric protons (complex carbohydrates), syringyl units (lignin), p-hydroxybenzoates (lignin). The resonance region from N-acetyl in peptidoglycan (PG) shows up in the HSQC spectrum for Clay #23 (Fig. 2a).

a


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b

Fig. 2 CMP 1H-13C HSQC spectra for Clay #23 (a) and Clay #21 (b) using DSMO-d6 solvent

The 2D 1H TOCSY spectrum for Clay #23 (Fig. 3a) displays the full 2D spectral range of microbial signatures as described by Simpson et al. 11, and peaks were assigned by comparison with the spectra of Fig. 18 and Fig. 2 in Simpson et al. 1 and Pautler et al. 26, respectively. The main resonance region is from albumin biopolymer,

1

or from microbial protein/peptide amino

acid side chains, 26 with a small portion of the resonance region around (1.5, 1.5) resulting from cuticle or microbial cell walls such as N-acetyl.

1

The region for amylopectin biopolymer

1

coincides with Region #2 centering around (3.5, 3.5) of Fig. 2D in Pautler et al. 26. Considering the bacterial interaction nature for Clay #23, this study agrees with Paulter et al. 26 that it results from microbial protein/peptide amino acid side chains (Fig. 3a) instead of amylopectin interpreted by Simpson et al. 1.



High resolution 1D and 2D NMR spectra such as HSQC, TOCSY can combinedly act as a detailed ‘fingerprint’ and profile of a wide range of metabolites in the samples (Figs. 1, 2 & 3). Besides the phenylalanine, tyrosine and uridine metabolites (Fig. 2a), some peaks in 1D and HSQC spectra of Clay #23 may be further assigned to specific metabolites based on Simpson et al. 1. The range from 1.6 to 1.9 ppm in 1D 1H spectra could be due to amino acid side chains; 1.5 ppm may be assigned to alanine (or CH2 γ to COOH); 1.0 ppm to valine; and 1.3 ppm could be assigned to (CH2)n or threonine. Owing to the complexity of the spectra and the much lower S/N for the spectra of most samples in this study, the exact identification of some metabolites was difficult, however, the aromatic region is highly resolved for Clay #23, permitting detailed assignments for this key region.

a


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b Fig. 3 CMP 1H TOCSY spectra for Clay #23 (a) and Clay #21 (b) using DSMO-d6 (specifically, red rectangle: microbial protein/peptide amino acid side chains; blue rectangle: cuticle or microbial cell walls such as N-Acetyl)

In summary, both the 1D and 2D CMP NMR spectra of Clay #23 reveal microbial signatures. The existence of N-acetyl, phenylalanine (aromatic) and tyrosine further confirm microbial interactions, as these OMs are not only from microbial bodies such as cuticle or cell wall, but also from the bio-products from microbial metabolites. The HSQC and TOCSY spectra from other clay samples, which had been processed with bacteria for shorter periods of time such as 1 day, only show aliphatic components (Fig. 2b and 3b) and weak aromatic resonances. This comparison is also echoed by the corresponding 1D 1H spectra. For these clay samples, the nutrition molecules and the preliminary metabolites had been



centrifuged and filtered out during the sample preparation due to their larger molecular sizes. Thus, both 1D and 2D NMR spectra for these samples display weaker and fewer resonance peaks, and indicate the lack of metabolites such as N-acetyl, phenylalanine, peptide/protein or albumin as well as carbohydrates. On the contrary, after interacting with bacteria for 3 days, the large nutrition molecules and preliminary metabolites had been consumed up by microbial interactions or centrifuged out, while many smaller metabolites had been produced in Clay #23, which result in the stronger NMR signals. 3.3. Conventional and CMP 1H-13C CPMAS NMR experiments for the bacteria-processed clay samples. Conventional 1H-13C CPMAS NMR experiments are commonly used to facilitate the polarization transfer from 1H to neighboring 13C through dipolar interactions in solids. Thus, CP is inefficient for solutions and gels, so that it acts as a filter to select only the most rigid components 14.

Due to the rigidity and heterogeneity of these bacteria-processed clay samples, the spectral

widths of the conventional CPMAS NMR resonances are relatively broad (Fig. 4), but it is still possible to assign these regions to the previously identified structural components: COO/NCO group at ~170 ppm, carbohydrate group centering ~70 ppm and an aliphatic group ~25 ppm (Fig. 4), giving an overview of the distribution of the carbon. Other much weaker but discernable 1H13C

resonances, such as 135 ppm (aromatic C), also exist for the conventional CPMAS NMR

spectra (Fig. 4). In this study, CMP was applied to obtain the 1H-13C CPMAS NMR spectra for all samples, but due to the low TOC in atmospheric particulates, only the bacteria-processed clay samples show resolvable CMP 13C CPMAS NMR signals. As a matter of fact, Clays #21 and #22 show three discernable CMP

13C

CPMAS spectral regions (Fig. 4), which are interpreted according to


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Simpson et al.

11:

the narrow peak area centering around 170 ppm is assigned as carbonyl, the

broader spectral region from 10 to 50 ppm with a very narrow peak at 30 ppm to aliphatics, and the last peak area covering from 50 to 80 ppm with very low S/N to carbohydrates. From the solution-state NMR spectra and data from previous studies, 1,11 the conventional and CMP

13C

CPMAS NMR spectra in this study can thus be divided into four major regions:

aliphatic, carbohydrate, aromatic, and carbonyl (Figs. 1 & 4). There are no clear spectral boundaries due to the overlap of these components. 1 The CMP 13C CPMAS spectra indicate that partial aliphatic and aromatic components (non-polar or hydrophobic) more likely occur as solid state in the solution, since only DSMO-d6 solvent can give discernible spectra (Fig. 4). DSMO-d6 may penetrate grain surfaces with these solved hydrophobic components. 11 Although Clay #23 has the strongest 1H NMR spectral signals with the highest resolution (Figs. 1, 2 and 3), it has few discernable CMP 13C CPMAS peaks, while Clay #21 and #22 have more discernable and stronger CMP 13C CPMAS resonances (Fig. 4). This is again accredited to metabolites caused by the intensive metabolism in Clay #23. Metabolites with small molecular size, such as glucose, amino acid side chains, acetate, alanine, threonine, lipoprotein and cutins, which are probably in a gel state adsorbed on surfaces, can significantly contribute to the resonances of solution 1H spectra, but not to CMP 13C CPMAS due to their lower rigidity and CP efficiency. In contrast, OMs with larger molecular size due to the insufficient metabolism in Clay #21 and 22, left after centrifuging preparation, may agglomerate together like a solid, giving the CMP 13C CPMAS peaks shown by Fig.4.



Fig. 4 The comparison between conventional (green) and CMP 1H-13C CPMAS spectra for Clay #21(red) and #22 (blue) in DMSO-d6

3.4. Interfacial dynamics for OM in clay samples processed with bacteria. Kinetic studies are very useful to explain surface properties such as sorption, chemical reactivity and degree of affinity for the adsorbents. CMP NMR can follow not only the free molecules but also the molecule’s penetration through the surfaces and sequestration into gel or solid phases. Surficial interactions and dynamics for different organic components in clay samples were here investigated by DE/TE CMP NMR experiments. (1) CMP 1H DE NMR experiments The signals from highly mobile components are suppressed by the DE NMR experiment, leaving only the signals from molecules with restricted diffusion such as protein biopolymers,


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cellulose and lipids, which are likely present in a gel-like (surficial) or states somewhere between a true liquid and solid. Based on the comparisons between 1D 1H and DE spectra (Figs. 1 and 5), most of the narrow signals in Fig. 1 are indeed suppressed, verifying that these resonances indeed arise from freely diffusing metabolites or small molecules. DE experiments usually represent surficial interactions such as adsorption,1 and such surficial affinity for these OMs can facilitate microbial interactions on layered surfaces within clays. Surficial adsorption usually works well for OMs with polarity, so D2O is often used as a solvent for CMP DE NMR experiment. Thus, the signals in the DE spectra, specifically using D2O, represent swollen molecules with restricted diffusion dominated by polar lipids and carbohydrates. Additionally, the comparisons among the 1H

CMP NMR spectra for Clay #23 (Figs 1, 2 and 5) indicate that aliphatics can be in solution,

surface, gel, or solid states, although Simpson et al. 1 showed that aliphatics preferentially bind to the surfaces (gel state). On the other hand, carbohydrates can be in both solution and gel states. Based on the dynamic or diffusional properties of the organic components and depending on solvents (DSMO-d6 or D2O) used, gel phases in this study were mainly determined to be from aliphatic components (non-polar for DSMO-d6 and polar for D2O), hydrocarbons (D2O), and trace amounts of aromatics (DSMO-d6). CMP 1H DE NMR spectra for Clay #21 and #22 have stronger peaks compared with Clay #23 (Fig. 5), which supports the interpretation made for CMP 13C CPMAS spectra. Therefore, due to lower microbial interactions compared to Clay #23, OM with larger molecular sizes in Clay #21 and #22 exists in a gel state and can even agglomerate together like solids.



Fig. 5 CMP 1H DE spectra for gel states in clay minerals processed with bacteria in D2O (Clay #23 to 20 sequentially listed from top to bottom) Note: carbohydrates show the strongest gel peak while there is almost no aromatic gel peak, but some high polar aliphatic components also are present as gels

Similarly, Simpson et al. 12 showed that a film swollen with water orients so that only the most polar functional groups from carbohydrates and carboxylic acids become exposed at the surface, while the more hydrophobic components such as aliphatic chains and aromatics are buried beneath the aqueous/film interface. Thus, surfaces within clays can be considered as important mediators for both hydrophobic and hydrophilic compounds. (2) CMP 1H T2-filtered NMR experiments


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The comparison between 1D 1H and TE spectra of Clay #23 in DMSO-D6 (Fig. 6) show the non-polarizable carbohydrates (from 3.2 to 3.75 ppm), with slower motion and larger sizes, are filtered out by TE. Thus, 1H TE spectra can provide another perspective to differentiate motional rates associated with molecular sizes.

Fig. 6 CMP 1D 1H (blue) and T2-Filtering (red) spectra for Clay #23 using DSMO-d6 solvent In summary, CMP NMR provides information not just about the structural categories present but also their molecular dynamics. These OMs, especially metabolites, can enter the space between the montmorillonite structural layers due to their small sizes, and even can expand these spaces. Furthermore, part of the metabolite molecules such as some aliphatic and aromatics can form gel



states on the surfaces, and even penetrate the layer surfaces along with DSMO-d6 giving rise to the resonance peaks of 13C CPMAS spectrum (Fig. 4). 3.5. Identification of OM components and their significance in samples of atmospheric particulates by CMP NMR. Due to the similarities in chemical and mineral compositions, environmental occurrences as well as the NMR spectra between atmospheric particulates and clay, the latter can serve as a model/reference and starting point for investigating atmospheric particulates. This study preliminarily identifies OMs and investigates the interaction between microorganisms and atmospheric particulates. This is also the main intent for this study, i.e., applying CMP NMR to probe the geochemical interaction mechanisms at a molecular/atomic scale for important environmental media such as atmospheric particulates and soil. Regardless of where they were collected, TOC in these samples of atmospheric particulates is

APPLICATIONS OF COMPREHENSIVE MULTIPHASE NMR TO

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