MS and NMR Analysis of Isotopically Labeled Chloramination Disinfection Byproducts: Hyperlinks and Chemical Reactions.

FT-ICR MS and NMR analysis of an isotopically labeled complex mixture of water disinfection byproducts formed by chloramine disinfection of model phenolic acids is described. A new molecular formula assignment procedure using the CoreMS Python library able to assign isotopically enriched formulas is proposed. Statistical analysis of the assigned formulas showed that the number of compounds, the diversity of the mixture, and the chlorine count increase during the chloramination reaction. The complex reaction mixture was investigated as a network of reactions using PageRank and Reverse PageRank algorithms. Independent of the MS signal intensities, the PageRank algorithm calculates the formulas with the highest probability at convergence of the reaction; these were chlorinated and nitrated derivatives of the starting materials. The Reverse PageRank revealed that the most probable chemical transformations in the complex mixture were chlorination and decarboxylation. These agree with the data obtained from INADEQUATE NMR spectra and literature data, indicating that this approach could be applied to gain insight into reactions pathways taking place in complex mixtures without any prior knowledge.

Responsible agriculture must adapt to the wetland character of mid-latitude peatlands.

Drained, lowland agricultural peatlands are greenhouse gas (GHG) emission hotspots and a large but vulnerable store of irrecoverable carbon. They exhibit soil loss rates of ~2.0 cm yr-1 and are estimated to account for 32% of global cropland emissions while producing only 1.1% of crop kilocalories. Carbon dioxide emissions account for >80% of their terrestrial GHG emissions and are largely controlled by water table depth. Reducing drainage depths is, therefore, essential for responsible peatland management. Peatland restoration can substantially reduce emissions. However, this may conflict with societal needs to maintain productive use, to protect food security and livelihoods. Wetland agriculture strategies will, therefore, be required to adapt agriculture to the wetland character of peatlands, and balance GHG mitigation against productivity, where halting emissions is not immediately possible. Paludiculture may substantially reduce GHG emissions but will not always be viable in the current economic landscape. Reduced drainage intensity systems may deliver partial reductions in the rate of emissions, with smaller modifications to existing systems. These compromise systems may face fewer hurdles to adoption and minimize environmental harm until societal conditions favour strategies that can halt emissions. Wetland agriculture will face agronomic, socio-economic and water management challenges, and careful implementation will be required. Diversity of values and priorities among stakeholders creates the potential for conflict. Successful implementation will require participatory research approaches and co-creation of workable solutions. Policymakers, private sector funders and researchers have key roles to play but adoption risks would fall predominantly on land managers. Development of a robust wetland agriculture paradigm is essential to deliver resilient production systems and wider environmental benefits. The challenge of responsible use presents an opportunity to rethink peatland management and create thriving, innovative and green wetland landscapes for everyone's future benefit, while making a vital contribution to global climate change mitigation.

High-Resolution Molecular-Level Characterization of a Blanket Bog Peat Profile.

To understand peatland carbon-cycling processes ultimately requires the ability to track changes occurring on the molecular-level. In this study, we profile a peat core taken from the world's largest blanket bog, Flow Country, Scotland, using physicochemical properties, ATR-FTIR, solid/liquid-state NMR, and solid/liquid-state FT-ICR-MS. Air-dried peat and labile and recalcitrant peat extracts, including pore water dissolved organic matter (PW-DOM), are analyzed and the merits of each technique are discussed. Solid-state NMR demonstrated changing distribution of compound classes with core depth and water table, the latter not picked up by IR. Liquid-state NMR and MS both demonstrated variations in molecular composition along the core depth in all phases and extracts. Contrary to previous reports, the composition of PW-DOM varied with depth. Major compounds, some previously unreported, identified by 1D/2D NMR occurred throughout the core, suggesting the existence of hot spots of microbial activity/compound accumulation. Offering complementary views, the techniques provided evidence of gradual molecular level changes with age, zonation due to the water table, and hot spots due to microbial activity. This study provides new insights into the molecular signatures of peat layers and establishes the foundation for examining peat function and health at the molecular-level.

Molecular Tagging for the Molecular Characterization of Natural Organic Matter.

Natural organic matter (NOM) is the product of microbial and abiotic decay of plant and animal remains in terrestrial and aquatic ecosystems. On a molecular level, NOM is a complex mixture of organic molecules, of which the vast majority of structures are unknown. By identifying these molecules, our understanding of the many functions of NOM could be greatly enhanced. However, given that they are chromatographically inseparable and number in the thousands, traditional analytical techniques have proven unable to achieve this goal. A promising approach to enumerate functional groups and elucidate molecular structures within NOM is based on a combination of molecular tagging and high resolution spectroscopic techniques, such as nuclear magnetic resonance spectroscopy and mass spectrometry. Molecular tagging involves the selective modification of particular functional groups, inserting nuclei to act as reporters on their surrounding chemical environment. This allows examination of only the tagged molecules within NOM, thereby reducing the complexity of the mixture. In this review, the effectiveness of molecular tagging methods incorporating carbon, silicon, nitrogen, phosphorus, and deuterium into NOM are discussed. Some potential tagging methods which have not yet been applied to NOM are also presented.

Reduced dimensionality hyphenated NMR experiments for the structure determination of compounds in mixtures.

For the structure determination of molecules in mixtures using NMR spectroscopy, the dispersion of 13C chemical shifts provides much needed separation of resonances in the indirectly detected dimension of 2D heterocorrelated NMR experiments. This separation is crucial for establishing networks of coupled spins by hyphenated techniques that combine hetero- and homonuclear polarisation transfers. However, as the sample complexity increases, 13C chemical shifts stop being unique, hindering spectral interpretation. The resulting ambiguities can be removed by adding another dimension to these experiments. However, the spectra obtained from complex samples are riddled with overlapped signals, meaning that another dimension will only reduce the spectral resolution and prevent structure determination. A promising solution is to stay in two dimensions and use the combined 13C and 1H chemical shifts to separate signals. We have developed a suite of (3,2)D reduced dimensionality hyphenated NMR experiments that preserve the information content of 3D spectra but offer all of the advantages of 2D spectra - high resolution and ease of manipulation with only a mild sensitivity penalty. The proposed experiments complement the existing (3,2)D HSQC-TOCSY and include a (3,2)D HSQC-NOESY/ROESY, (3,2)D HSQC-CLIP-COSY and (3,2)D HSQC-HSQMBC. The new experiments represent a set of NMR techniques typically employed in the structure determination of complex compounds and have been adopted here for use on mixtures. The resolving power of these experiments is illustrated on the analysis of hot water extracts of green tea.

Advanced solvent signal suppression for the acquisition of 1D and 2D NMR spectra of Scotch Whisky.

A simple and robust solvent suppression technique that enables acquisition of high-quality 1D 1 H nuclear magnetic resonance (NMR) spectra of alcoholic beverages on cryoprobe instruments was developed and applied to acquire NMR spectra of Scotch Whisky. The method uses 3 channels to suppress signals of water and ethanol, including those of 13 C satellites of ethanol. It is executed in automation allowing high throughput investigations of alcoholic beverages. On the basis of the well-established 1D nuclear Overhauser spectroscopy (NOESY) solvent suppression technique, this method suppresses the solvent at the beginning of the pulse sequence, producing pure phase signals minimally affected by the relaxation. The developed solvent suppression procedure was integrated into several homocorrelated and heterocorrelated 2D NMR experiments, including 2D correlation spectroscopy (COSY), 2D total correlation spectroscopy (TOCSY), 2D band-selective TOCSY, 2D J-resolved spectroscopy, 2D 1 H, 13 C heteronuclear single-quantum correlation spectroscopy (HSQC), 2D 1 H, 13 C HSQC-TOCSY, and 2D 1 H, 13 C heteronuclear multiple-bond correlation spectroscopy (HMBC). A 1D chemical-shift-selective TOCSY experiments was also modified. The wealth of information obtained by these experiments will assist in NMR structure elucidation of Scotch Whisky congeners and generally the composition of alcoholic beverages at the molecular level.

Isotope-Filtered 4D NMR Spectroscopy for Structure Determination of Humic Substances.

Humic substances, the main component of soil organic matter, could form an integral part of green and sustainable solutions to the soil fertility problem. However, their global-scale application is hindered from both scientific and regulatory perspectives by the lack of understanding of the molecular make-up of these chromatographically inseparable mixtures containing thousands of molecules. Here we show how multidimensional NMR spectroscopy of isotopically tagged molecules enables structure characterization of humic compounds. We illustrate this approach by identifying major substitution patterns of phenolic aromatic moieties of a peat soil fulvic acid, an operational fraction of humic substances. Our methodology represents a paradigm shift in the use of NMR active tags in structure determination of small molecules in complex mixtures. Unlike previous tagging methodologies that focused on the signals of the tags, we utilize tags to directly probe the identity of the molecules they are attached to.

Detecting low-level flexibility using residual dipolar couplings: a study of the conformation of cellobiose.

We have developed novel NMR methods for the measurement of heteronuclear residual dipolar couplings (RDCs) in molecules with severely overlapping NMR resonances. These and other methods enabled us to obtain 31 RDCs for α-D-cellobiose and 24 RDCs for ß-D-cellobiose. The interpretation of the data in the approximation of a rigid disaccharide structure, using RDCs and interglycosidic (3)J coupling constants, yielded conformation that is very close to that determined using X-ray crystallography. However, depending on which ring was used to calculate the order parameters, the dihedral angle ψH varied up to 30° or 40°, while the φH angle was always the same. This indicates residual flexibility of the glycosidic linkage between the two monosaccharide rings and was observed for both α- and ß-D-cellobiose. The RDC analysis using rigid fragments rather than a complete molecule has thus shown that the glycosidic bond of cellobiose is not completely rigid and exhibits low-level flexibility. The sources of this flexibility are discussed and evidence presented to support a hypothesis that it is associated with the ψ more than the φ angle.

New 19F NMR methodology reveals structures of molecules in complex mixtures of fluorinated compounds.

Although the number of natural fluorinated compounds is very small, fluorinated pharmaceuticals and agrochemicals are numerous. 19F NMR spectroscopy has a great potential for the structure elucidation of fluorinated organic molecules, starting with their production by chemical or chemoenzymatic reactions, through monitoring their structural integrity, to their biotic and abiotic transformation and ultimate degradation in the environment. Additionally, choosing to incorporate 19F into any organic molecule opens a convenient route to study reaction mechanisms and kinetics. Addressing limitations of the existing 19F NMR techniques, we have developed methodology that uses 19F as a powerful spectroscopic spy to study mixtures of fluorinated molecules. The proposed 19F-centred NMR analysis utilises the substantial resolution and sensitivity of 19F to obtain a large number of NMR parameters, which enable structure determination of fluorinated compounds without the need for their separation or the use of standards. Here we illustrate the 19F-centred structure determination process and demonstrate its power by successfully elucidating the structures of chloramination disinfectant by-products of a single mono-fluorinated phenolic compound, which would have been impossible otherwise. This novel NMR approach for the structure elucidation of molecules in complex mixtures represents a major contribution towards the analysis of chemical and biological processes involving fluorinated compounds.

19F-centred NMR analysis of mono-fluorinated compounds.

Addressing limitations of the existing NMR techniques for the structure determination of mono-fluorinated compounds, we have developed methodology that uses 19F as the focal point of this process. The proposed 19F-centred NMR analysis consists of a complementary set of broadband, phase-sensitive NMR experiments that utilise the substantial sensitivity of 19F and its far reaching couplings with 1H and 13C to obtain a large number of NMR parameters. The assembled 1H, 13C and 19F chemical shifts, values of J HF, J HH, and J FC coupling constants and the size of 13C induced 19F isotopic shifts constitute a rich source of information that enables structure elucidation of fluorinated moieties and even complete structures of molecules. Here we introduce the methodology, provide a detailed description of each NMR experiment and illustrate their interpretation using 3-fluoro-3-deoxy-d-glucose. This novel approach performs particularly well in the structure elucidation of fluorinated compounds embedded in complex mixtures, eliminating the need for compound separation or use of standards to confirm the structures. It represents a major contribution towards the analysis of fluorinated agrochemicals and (radio)pharmaceuticals at any point during their lifetime, including preparation, use, biotransformation and biodegradation in the environment. The developed methodology can also assist with the investigations of the stability of fluoroorganics and their pharmacokinetics. Studies of reaction mechanisms using fluorinated molecules as convenient reporters of these processes, will also benefit.

PyKrev: A Python Library for the Analysis of Complex Mixture FT-MS Data.

In this paper, we present PyKrev, a Python library for the analysis of complex mixture Fourier transform mass spectrometry (FT-MS) data. PyKrev is a comprehensive suite of tools for analysis and visualization of FT-MS data after formula assignment has been performed. These comprise formula manipulation and calculation of chemical properties, intersection analysis between multiple lists of formulas, calculation of chemical diversity, assignment of compound classes to formulas, multivariate analysis, and a variety of visualization tools producing van Krevelen diagrams, class histograms, PCA score, and loading plots, biplots, scree plots, and UpSet plots. The library is showcased through analysis of hot water green tea extracts and Scotch whisky FT-ion cyclotron resonance-MS data sets. PyKrev addresses the lack of a single, cohesive toolset for researchers to perform FT-MS analysis in the Python programming environment encompassing the most recent data analysis techniques used in the field.

Towards a microbial process-based understanding of the resilience of peatland ecosystem service provisioning - A research agenda.

Peatlands are wetland ecosystems with great significance as natural habitats and as major global carbon stores. They have been subject to widespread exploitation and degradation with resulting losses in characteristic biota and ecosystem functions such as climate regulation. More recently, large-scale programmes have been established to restore peatland ecosystems and the various services they provide to society. Despite significant progress in peatland science and restoration practice, we lack a process-based understanding of how soil microbiota influence peatland functioning and mediate the resilience and recovery of ecosystem services, to perturbations associated with land use and climate change. We argue that there is a need to: in the short-term, characterise peatland microbial communities across a range of spatial and temporal scales and develop an improved understanding of the links between peatland habitat, ecological functions and microbial processes; in the medium term, define what a successfully restored 'target' peatland microbiome looks like for key carbon cycle related ecosystem services and develop microbial-based monitoring tools for assessing restoration needs; and in the longer term, to use this knowledge to influence restoration practices and assess progress on the trajectory towards 'intact' peatland status. Rapid advances in genetic characterisation of the structure and functions of microbial communities offer the potential for transformative progress in these areas, but the scale and speed of methodological and conceptual advances in studying ecosystem functions is a challenge for peatland scientists. Advances in this area require multidisciplinary collaborations between peatland scientists, data scientists and microbiologists and ultimately, collaboration with the modelling community. Developing a process-based understanding of the resilience and recovery of peatlands to perturbations, such as climate extremes, fires, and drainage, will be key to meeting climate targets and delivering ecosystem services cost effectively.

The Response of Microbial Communities to Peatland Drainage and Rewetting. A Review.

Peatlands are significant global carbon stores and play an important role in mediating the flux of greenhouse gasses into the atmosphere. During the 20th century substantial areas of northern peatlands were drained to repurpose the land for industrial or agricultural use. Drained peatlands have dysfunctional microbial communities, which can lead to net carbon emissions. Rewetting of drained peatlands is therefore an environmental priority, yet our understanding of the effects of peatland drainage and rewetting on microbial communities is still incomplete. Here we summarize the last decade of research into the response of the wider microbial community, methane-cycling microorganisms, and micro-fauna to drainage and rewetting in fens and bogs in Europe and North America. Emphasis is placed on current research methodologies and their limitations. We propose targets for future work including: accounting for timescale of drainage and rewetting events; better vertical and lateral coverage of samples across a peatland; the integration of proteomic and metabolomic datasets into functional community analysis; the use of RNA sequencing to differentiate the active community from legacy DNA; and further study into the response of the viral and micro-faunal communities to peatland drainage and rewetting. This review should benefit researchers embarking on studies in wetland microbiology and non-microbiologists working on peatland drainage and rewetting in general.

Molecular level study of hot water extracted green tea buried in soils - a proxy for labile soil organic matter.

Understanding the composition of soil organic matter (SOM) is vital to our understanding of how soils form, evolve and respond to external stimuli. The shear complexity of SOM, an inseparable mixture of thousands of compounds hinders the determination of structure-function relationships required to explore these processes on a molecular level. Litter bags and soil hot water extracts (HWE) have frequently been used to study the transformation of labile SOM, however these are still too complex to examine beyond compound classes. In this work, a much simpler mixture, HWE buried green tea, was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS), as a proxy for labile SOM. Changes induced by the burial over 90 days in a grassland, woodland and two peatland sites, one damaged by drainage and one undergoing restoration by drain-blocking, were analysed. Major differences between the extracts were observed on the level of compound classes, molecular formulae and specific molecules. The causes of these differences are discussed with reference to abiotic and biotic processes. Despite the vastly different detection limits of NMR and MS, chemometric analysis of the data yielded identical separation of the samples. These findings provide a basis for the molecular level interrogation of labile SOM and C-cycling processes in soils.

NMR methodology for complex mixture 'separation'.

Mixture 'separation' by NMR is demonstrated through the development of a pseudo 4D NMR experiment, 3D IPAP INEPT-INADEQUATE-HSQC, designed for the structural elucidation of (13)C tagged compounds.