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The soil carbon (C) saturation concept suggests an upper limit to the storage of soil organic carbon (SOC). It is set by the mechanisms that protect soil organic matter from mineralization. Biochar has the capacity to protect new C, including rhizodeposits and microbial necromass. However, the decadal-scale mechanisms by which biochar influences the molecular diversity, spatial heterogeneity, and temporal changes in SOC persistence, remain unresolved. Here we show that the soil C storage ceiling of a Ferralsol under subtropical pasture was raised by a second application of Eucalyptus saligna biochar 8.2 years after the first application—the first application raised the soil C storage ceiling by 9.3 Mg new C ha−1 and the second application raised this by another 2.3 Mg new C ha−1. Linking direct visual evidence from one-, two-, and three-dimensional analyses with SOC quantification, we found high spatial heterogeneity of C functional groups that resulted in the retention of rhizodeposits and microbial necromass in microaggregates (53–250 µm) and the mineral fraction (

Refereed/Peer-reviewed Soil organic carbon management is a nature-based carbon dioxide removal technology at the same time contributing to soil health and agricultural productivity. The soil science communities are refuting the traditional assumptions of the nature of soil organic matter (SOM) as based on 'humic substances' that are operationally-defined and have not been observed by contemporary, in situ spectromicroscopic techniques. Instead, new theories suggest that the interactions between molecular diversity of organic compounds, their spatial heterogeneity and temporal variability controls the formation and persistence of SOM. A mechanistic understanding of these processes occurring within organo-mineral and organo-organic assemblages requires non-invasive techniques that minimize any disturbance to the physical and chemical integrity of the sample. Here, we present a theory-driven review where a combination of in situ methods serve as potential solutions to better understand the persistence and dynamics of SOM and its effects on nutrient distribution at a micro- and nano-scale. We explore underlying theories in light of advances in available methodologies, their historical development and future opportunities. Examples of interdisciplinary approaches that have been utilized in other areas of science but not in soils offer both deductive and inductive analytical opportunities. We show how different conceptual methods across scales inform each other, and how important and indispensable high-resolution investigations are to resolving next-generation questions.

The Infrared Microspectroscopy Beamline at the Australian Synchrotron is equipped with a Fourier transform infrared (FTIR) spectrometer, which is coupled with an infrared (IR) microscope and a choice of two detectors: a single-point narrow-band mercury cadmium telluride (MCT) detector and a 64 × 64 multi-pixel focal plane array (FPA) imaging detector. A scanning-based point-by-point mapping method is commonly used with a tightly focused synchrotron IR beam at the sample plane, using an MCT detector and a matching 36× IR reflecting objective and condenser (NA = 0.5), which is time consuming. In this study, the beam size at the sample plane was increased using a 15× objective and the spatio-spectral aberrations were investigated. A correlation-based semi-synthetic computational optical approach was applied to assess the possibilities of exploiting the aberrations to perform rapid imaging rather than a mapping approach.

Biological recycling of inorganic phosphorus (Pi) from organic phosphorus (Po) compounds by phosphatase-type enzymes, including phytases, is an important contributor to the pool of bioavailable P to plants and microorganisms. However, studies of mixed-substrate reactions with these enzymes are lacking. Here, we explore the reactivity of a phytase extract from the fungus Aspergillus niger toward a heterogeneous mixture containing, in addition to phytate, different structures of environmentally relevant Po compounds such as ribonucleotides and sugar phosphates. Using a high-resolution liquid chromatography-mass spectrometry method to monitor simultaneously the parent Po compounds and their by-products, we captured sequential substrate-specific evolution of Pi from the mixture, with faster hydrolysis of multiphosphorylated compounds (phytate, diphosphorylated sugars, and di- and tri-phosphorylated ribonucleotides) than hydrolysis of monophosphorylated compounds (monophosphorylated sugars and monophosphorylated ribonucleotides). The interaction mechanisms and energies revealed by molecular docking simulations of each Po compound within the enzyme's active site explained the substrate hierarchy observed experimentally. Specifically, the favorable orientation for binding of the negatively charged phosphate moieties with respect to the positive potential surface of the active site was important. Collectively, our findings provide mechanistic insights about the broad but hierarchical role of phytase-type enzymes in Pi recycling from the heterogeneous assembly of Po compounds in agricultural soils or wastes.

The quantification of amino acids as freely dissolved compounds or from hydrolyzed peptides and proteins is a common endeavor in biomedical and environmental sciences. In order to avoid the drawbacks of derivatization and application challenges of tandem mass spectrometry, we present here a robust 13-min liquid chromatography coupled with a full-scan mass spectrometry method to achieve rapid detection and quantification of 30 amino acids without derivatization. We combined hydrophilic interaction liquid chromatography with heated electrospray ionization and high-resolution mass spectrometry operated with polarity switching to analyze the 20 proteinogenic amino acids, ornithine, citrulline, homoserine, cystine, and six acetylated amino acids. We obtained high mass accuracy and good precision of the targeted amino acids. Limits of detection ranged from 0.017 to 1.3 µM. Noteworthy for environmental samples, we found comparable ionization efficiency and quantitative detection for the majority of the analytes prepared with pure water versus a high-salt solution. We applied the method to profile carbon source-dependent secretions of amino acids by Pseudomonas protegens Pf-5, a well-known plant-beneficial bacterium.

Iron (Fe) (oxyhydr)oxide minerals, which are amongst most reactive minerals in soils and sediments, are known to exhibit strong adsorption of inorganic phosphate (Pi) and organophosphate (Po) compounds. Beyond synthetic Po compounds, much still remains unknown about the reactivity of these minerals to transform naturally-occurring Po compounds to Pi, particularly with respect to solution versus surface speciation of Po hydrolysis. To investigate this reactivity with a ferrihydrite-type mineral and ribonucleotides, we employed high-resolution liquid chromatography-mass spectrometry (LC-MS), X-ray absorption near-edge structure (XANES), Fourier-transform infrared (FTIR) spectroscopy, and molecular modeling. Kinetic experiments were conducted with the mineral (1 g L−1) reacted with adenosine monophosphate, diphosphate, or triphosphate (respectively AMP, ADP, ATP; 50 µM). Analysis of solution organic species by LC-MS implied that only adsorption occurred with AMP and ADP but both adsorption and dephosphorylation of ATP were evident. Maximum adsorption capacities per gram of mineral were 40.6 ± 0.8 µmol AMP, 35.7 ± 1.6 µmol ADP, and 10.9 ± 1.0 µmol ATP; solution dephosphorylated by-products accounted for 15% of initial ATP. Subsequent XANES analysis of the surface species revealed that 16% of adsorbed AMP and 30% of adsorbed ATP were subjected to dephosphorylation, which was not fully quantifiable from the solution measurements. Molecular simulations predicted that ADP and ATP were complexed mainly via the phosphate groups whereas AMP binding also involved multiple hydrogen bonds with the adenosine moiety; our FTIR data confirmed these binding confirmations. Our findings thus imply that specific adsorption mechanisms dictate the recycling and subsequent trapping of Pi from ribonucleotide-like biomolecules reacted with Fe (oxyhydr)oxide minerals.

Atrazine and nitrate NO 3 - N are two agricultural pollutants that occur widely in surface and groundwater. One of the pathways by which these pollutants reach surface water is through subsurface drainage tile lines. Edge-of-field anaerobic denitrifying bioreactors apply organic substrates such as woodchips to stimulate the removal of NO 3 - N from the subsurface tile waters through denitrification. Here we investigated the co-removal of NO 3 − N and atrazine by these bioreactors. Laboratory experiments were conducted using 12-L woodchips-containing flow-through bioreactors, with and without the addition of biochar, to treat two concentrations of atrazine (20 and 50 μg L−1) and NO 3 − N (1.5 and 11.5 mg L−1), operated at four hydraulic retention time, HRT, (4 h, 8 h, 24 h, 72 h). Additionally, we examined the effect of aerating the bioreactors on atrazine removal. Furthermore, we tested atrazine removal by a field woodchip denitrifying bioreactor. The removal of both NO 3 − N and atrazine increased with increasing HRT in the laboratory bioreactors. At 4 h, the woodchip bioreactors removed 65% of NO 3 − N and 25% of atrazine but, at 72 h, the bioreactors eliminated all the NO 3 − N and 53% of atrazine. Biochar-amended bioreactors removed up to 90% of atrazine at 72-h retention time. We concluded that atrazine removal was primarily via adsorption because neither aeration nor NO 3 − N levels had an effect. At 4-h retention time, the field bioreactors achieved 2.5 times greater atrazine removal than the laboratory bioreactors. Our findings thus highlighted hydraulic retention time and biochar amendments as two important factors that may control the efficiency of atrazine removal by denitrifying bioreactors. In sum, laboratory and field data demonstrated that denitrifying bioreactors have the potential to decrease pesticide transport from agricultural lands to surface waters.

Analysis of the epicuticular wax layer on the surface of plant leaves can provide a unique window into plant physiology and responses to environmental stimuli. Well‐established analytical methodologies can quantify epicuticular wax composition, yet few methods are capable of imaging wax distribution in situ or in vivo. Here, the first report of Fourier transform infrared (FTIR) reflectance spectroscopic imaging as a non‐destructive, in situ, method to investigate variation in epicuticular wax distribution at 25 µm spatial resolution is presented. The authors demonstrate in vivo imaging of alterations in epicuticular waxes during leaf development and in situ imaging during plant disease or exposure to environmental stressors. It is envisaged that this new analytical capability will enable in vivo studies of plants to provide insights into how the physiology of plants and crops respond to environmental stresses such as disease, soil contamination, drought, soil acidity, and climate change., Fourier transform infrared (FTIR) reflectance spectroscopic imaging has been used to image epicuticular waxes on the surface of plant leaves, in living plants, to observe how plant physiology changes in response to environmental stressors or disease.

Infrared (IR) microspectroscopy is a powerful molecular fingerprinting tool widely used for the identification of structural and functional composition of biological and chemical samples. The IR microspectroscopy beamline at the Australian Synchrotron can be operated either with a single-point narrow-band mercury cadmium telluride (MCT) detector or a focal plane array (FPA) imaging detector with 64 × 64 pixels. For the implementation of indirect nonscanning imaging technology, the system was operated with the FPA detector. In this study, we propose an indirect IR imaging technique based on the principles of correlation optics using diffractive optical elements such as random pinhole array (RPA) and Fresnel zone plate (FZP). The spatial and spectral variations of point spread functions (PSFs) of the RPA and FZP were simulated for the synchrotron configuration. Intensity responses for 2D objects were simulated using the same simulation conditions and reconstructed using Lucy-Richardson algorithm. Fabrication of diffractive elements for IR wavelengths is often a challenging task as the IR transparent material substrates, such as barium fluoride and calcium fluoride, are highly susceptible to thermal shocks and brittle by nature. The diffractive elements were fabricated by ablating directly on a 100 nm thick gold coated substrate using femtosecond laser pulses. The simulation results and the fabrication outcomes demonstrate the feasibility of indirect imaging at the synchrotron IR beamline.

Biological recycling of inorganic phosphorus (P

We used time-resolved metabolic footprinting, an important technical approach used to monitor changes in extracellular compound concentrations during microbial growth, to study the order of substrate utilization (i.e., substrate preferences) and kinetics of a fast-growing soil isolate, Paraburkholderia sp. strain 1N. The growth of Paraburkholderia sp. 1N was monitored under aerobic conditions in a soil-extracted solubilized organic matter medium, representing a realistic diversity of available substrates and gradient of initial concentrations. We combined multiple analytical approaches to track over 150 compounds in the medium and complemented this with bulk carbon and nitrogen measurements, allowing estimates of carbon use efficiency throughout the growth curve. Targeted methods allowed the quantification of common low-molecular-weight substrates: glucose, 20 amino acids, and 9 organic acids. All targeted compounds were depleted from the medium, and depletion followed a sigmoidal curve where sufficient data were available. Substrates were utilized in at least three distinct temporal clusters as Paraburkholderia sp. 1N produced biomass at a cumulative carbon use efficiency of 0.43. The two substrates with highest initial concentrations, glucose and valine, exhibited longer usage windows, at higher biomass-normalized rates, and later in the growth curve. Contrary to hypotheses based on previous studies, we found no clear relationship between substrate nominal oxidation state of carbon (NOSC) or maximal growth rate and the order of substrate depletion. Under soil solution conditions, the growth of Paraburkholderia sp. 1N induced multiauxic substrate depletion patterns that could not be explained by the traditional paradigm of catabolite repression. IMPORTANCE Exometabolomic footprinting methods have the capability to provide time-resolved observations of the uptake and release of hundreds of compounds during microbial growth. Of particular interest is microbial phenotyping under environmentally relevant soil conditions, consisting of relatively low concentrations and modeling pulse input events. Here, we show that growth of a bacterial soil isolate, Paraburkholderia sp. 1N, on a dilute soil extract resulted in a multiauxic metabolic response, characterized by discrete temporal clusters of substrate depletion and metabolite production. Our data did not support the hypothesis that compounds with lower energy content are used preferentially, as each cluster contained compounds with a range of nominal oxidation states of carbon. These new findings with Paraburkholderia sp. 1N, which belongs to a metabolically diverse genus, provide insights on ecological strategies employed by aerobic heterotrophs competing for low-molecular-weight substrates in soil solution.

Exometabolomic footprinting methods have the capability to provide time-resolved observations of the uptake and release of hundreds of compounds during microbial growth. Of particular interest is microbial phenotyping under environmentally relevant soil conditions, consisting of relatively low concentrations and modeling pulse input events. Here, we show that growth of a bacterial soil isolate, Paraburkholderia sp. 1N, on a dilute soil extract resulted in a multiauxic metabolic response, characterized by discrete temporal clusters of substrate depletion and metabolite production. Our data did not support the hypothesis that compounds with lower energy content are used preferentially, as each cluster contained compounds with a range of nominal oxidation states of carbon. These new findings with Paraburkholderia sp. 1N, which belongs to a metabolically diverse genus, provide insights on ecological strategies employed by aerobic heterotrophs competing for low-molecular-weight substrates in soil solution., We used time-resolved metabolic footprinting, an important technical approach used to monitor changes in extracellular compound concentrations during microbial growth, to study the order of substrate utilization (i.e., substrate preferences) and kinetics of a fast-growing soil isolate, Paraburkholderia sp. strain 1N. The growth of Paraburkholderia sp. 1N was monitored under aerobic conditions in a soil-extracted solubilized organic matter medium, representing a realistic diversity of available substrates and gradient of initial concentrations. We combined multiple analytical approaches to track over 150 compounds in the medium and complemented this with bulk carbon and nitrogen measurements, allowing estimates of carbon use efficiency throughout the growth curve. Targeted methods allowed the quantification of common low-molecular-weight substrates: glucose, 20 amino acids, and 9 organic acids. All targeted compounds were depleted from the medium, and depletion followed a sigmoidal curve where sufficient data were available. Substrates were utilized in at least three distinct temporal clusters as Paraburkholderia sp. 1N produced biomass at a cumulative carbon use efficiency of 0.43. The two substrates with highest initial concentrations, glucose and valine, exhibited longer usage windows, at higher biomass-normalized rates, and later in the growth curve. Contrary to hypotheses based on previous studies, we found no clear relationship between substrate nominal oxidation state of carbon (NOSC) or maximal growth rate and the order of substrate depletion. Under soil solution conditions, the growth of Paraburkholderia sp. 1N induced multiauxic substrate depletion patterns that could not be explained by the traditional paradigm of catabolite repression. IMPORTANCE Exometabolomic footprinting methods have the capability to provide time-resolved observations of the uptake and release of hundreds of compounds during microbial growth. Of particular interest is microbial phenotyping under environmentally relevant soil conditions, consisting of relatively low concentrations and modeling pulse input events. Here, we show that growth of a bacterial soil isolate, Paraburkholderia sp. 1N, on a dilute soil extract resulted in a multiauxic metabolic response, characterized by discrete temporal clusters of substrate depletion and metabolite production. Our data did not support the hypothesis that compounds with lower energy content are used preferentially, as each cluster contained compounds with a range of nominal oxidation states of carbon. These new findings with Paraburkholderia sp. 1N, which belongs to a metabolically diverse genus, provide insights on ecological strategies employed by aerobic heterotrophs competing for low-molecular-weight substrates in soil solution.

Microbial processing of fresh carbon inputs is recognized as a key step in the formation of mineral-associated organic matter. Low molecular weight (LMW) compounds comprise a notable fraction of th...

Dissolved natural organic matter (DNOM) is primarily composed of exuded or remnant biomolecules from plants and microorganisms. Labile amino acids and sugars have been well documented in the low molecular weight components of DNOM. However, little attention has been devoted to the isolation of labile short-chain carboxylic acids (SSCAs), which have demonstrated biogeochemical significance as sources of assimilable carbon, promoters of mineral dissolution, and ligands for metal complexation. Here we present an analytical method that identifies compound-specific SSCAs in a peat-derived DNOM isolate, Pahokee peat humic acid. Using high-resolution liquid chromatography (LC) coupled with high-accurate orbitrap mass spectrometry (MS), we targeted the identification of five SSCAs of different types: gluconate (a C6 monocarboxylic acid), citrate (a branched C6 tricarboxylic acid), 2-ketoglutarate (a C5 dicarboxylic acid), and malate and fumarate (C4 dicarboxylic acids). Following LC-separation and electrospray ionization, the compounds were annotated directly by orbitrap MS using their exact mass-over-charge (m/z) ions in the negative mode and their stoichiometric composition. Validated by LC–MS metabolite annotation in a bacterial matrix, we achieved identification of all five compounds in the peat DNOM isolate. Each targeted m/z channel also captured non-targeted compounds at different retention times, which represent isomers or different compounds. We found that the five targeted and the two non-targeted SSCAs identified collectively accounted for high parts-per-million to low parts-per-thousand of the total carbon, oxygen, or carboxyl content. Building on these findings, an important next step is to obtain a comprehensive profiling of SSCA structures in DNOM of different origins.

Microcystins are toxic cyclic peptides produced worldwide by cyanobacteria in surface caters. The general structure of microcystin is inherently amenable to metal complexation. However, structural characterization of metal–microcystin complexes is lacking. Here we performed molecular dynamics simulations to obtain structures of aqueous complexes of microcystin–leucine–arginine and microcystin–arginine–arginine with Ca2+, Mg2+, Fe2+, Zn2+, and Cu2+. Results show that complexes with Cu2+ and Zn2+ were the most stable. Shorter metal-O atom distances result in more favorable complexes. For instance, the relatively stronger Zn–microcystin complexes have metal-Ocarboxyl distances of 1.78 A, whereas the weaker Ca–microcystin complexes have this distance greater than 2.0 A. Favorable metal complexation is attributed to the conformation of the microcystin peptide cavity that facilitated a specific coordination geometry of carboxyl and keto O atoms around the metal cation. Our findings imply that the cellular and extracellular roles of microcystin with respect to metal chelation are controlled both by the metal species and by the population of microcystin variants.

Soil C is a key factor influencing soil health and, by inference, ecosystem condition. In arid and semi-arid regions, soil moisture limits accumulation and decomposition of soil C. On floodplains, soil moisture can come from rainfall events or periodic flooding. The effect of flooding on soil C decomposition has received some attention in the literature but mostly through the study of agricultural systems or mesocosms. Field studies of actual floods are not common. We measured soil C response to a managed flood on a semi-arid lowland river floodplain in southeastern Australia before and after inundation at control (unflooded), floodplain (low hydraulic energy), and flood runner (high hydraulic energy) sites. At control sites, soil C changed little from before to 16 d after the onset of the flood. At flooded sites, most measures of soil C (total soil organic matter [floodplain sites only], permanganate-oxidizable C, persulfate-extractable C, water-extractable C [flood runner sites only], microbial ...

Environmental contextSphagnum moss is a widespread species in peatlands globally and responsible for a large fraction of carbon storage in these systems. We used synchrotron infrared microspectroscopy to characterise the acid-base properties of Sphagnum moss and the conditions under which calcium uptake can occur (essential for plant tissue integrity). The work allows a chemical model for Sphagnum distribution in the landscape to be proposed. AbstractSphagnum is one the major moss types responsible for the deposition of organic soils in peatland systems. The cell walls of this moss have a high proportion of carboxylated polysaccharides (polygalacturonic acids), which act as ion exchangers and are likely to be important for the structural integrity of the cell walls. We used synchrotron light source infrared microspectroscopy to characterise the acid-base and calcium complexation properties of the cell walls of Sphagnum cristatum stems, using freshly sectioned tissue confined in a flowing liquid cell with both normal water and D2O media. The Fourier transform infrared spectra of acid and base forms are consistent with those expected for protonated and deprotonated aliphatic carboxylic acids (such as uronic acids). Spectral deconvolution shows that the dominant aliphatic carboxylic groups in this material behave as a monoprotic acid (pKa=4.97–6.04). The cell wall material shows a high affinity for calcium, with a binding constant (K) in the range 103.9–104.7 (1:1 complex). The chemical complexation model developed here allows for the prediction of the chemical environment (e.g. pH, ionic content) under which Ca2+ uptake can occur, and provides an improved understanding for the observed distribution of Sphagnum in the landscape.

The redox properties of Fe(II) adsorbed onto mineral surfaces have been highly studied over recent years due to the wide range of environmental contaminants that react with this species via abiotic processes. In this work the reactivity of Fe(II) adsorbed onto hydrous ferric oxide (HFO) has been studied using ferrocene (bis-cyclopentadienyl iron(II); Fc) derivatives as electron shuttles in cyclic voltammetry (CV) experiments. The observed amplification of the ferrocene oxidation peak in CV is attributed to reaction between the electrochemically generated ferrocenium (Fc(+)) ion and adsorbed Fe(II) species in a catalytic process (EC' mechanism). pH dependence studies show that the reaction rate increases with Fe(II) adsorption and is maintained in the absence of aqueous Fe(2+), providing strong evidence that the electron transfer process involves the adsorbed species. The rate of reaction between Fc(+) and adsorbed Fe(II) increases with the redox potential of the ferrocene derivative, as expected, with bimolecular rate constants in the range 10(3)-10(5) M(-1) s(-1). The ferrocene-mediated electrochemical method described has considerable promise in the development of a technique for measuring electron-transfer rates in geochemical and environmental systems.

MDFRC item.Microcystins (MCs) are a group of hepatotoxins produced by cyanobacteria that have not had their functional role or the environmental factors that trigger production clearly determined. One suggestion is that microcystins are siderophores (i.e., ligands with an extremely high affinity with iron, typically with stability constants substantially greater than 10²⁵). In this work, we explore proton and iron binding with microcystin-LR (MC-LR). Using UV−visible spectroscopy and a HPLC peak retention time-based method, the two acid dissociation constants associated with the carboxylic groups of MC-LR were determined to be: pKa1 = 2.17 and pKa2 = 3.96. Cyclic voltammetry provides evidence for the formation of at least two FeIII-MC-LR complexes, with the FeIII reduction peak significantly shifted to more reducing potentials in the presence of MC-LR. These complexes have been interpreted as a rapidly formed initial complex (Complex 1) and a more stable, and slower forming, Complex 2. The stability constant for FeIII-MC-LR (Complex 2) was estimated to be approximately 10¹³ in 60% v/v MeOH/water at 0.1 M ionic strength. The electrochemical experiments provide no evidence for the formation of a complex between Fe2+ and MC-LR. Given that most MC-LR is released only upon cell lysis, and coupled with the moderate strength of the stability constant with FeIII determined in this study, it appears unlikely that that MC-LR is an extracellular siderophore. If MC-LR is involved in iron regulation in cyanobacteria, it is more likely as a shuttle for iron across the cell membrane or in intracellular processes.

Environmental context Acidification of inland waterways is an emerging issue worldwide, mostly because it disturbs the balance of reduced sulfur species in soils, sediments and mine tailings. We describe a pathway for wetland acidification through salt displacement and oxidation of Fe2+ from clay minerals. This alternative pathway for acidification raises environmental concerns because an increasing number of inland waterways are affected by increasing salinity. Abstract A wetland near the Murray River (south-eastern Australia) was found to have significant levels of exchangeable reduced iron (Fe2+) in the sediment clay-zone, and the potential for acidification under high salinity and oxidising conditions. Cation exchange experiments using purified clay from this site show relative affinities consistent with the lyotrophic series: Fe2+>Ca2+>Mg2+>H+>K+>Na+. This relative affinity is confirmed in Fe2+ displacement experiments using natural sediment clay. Proton production during oxidation of salt-treated sediments corresponds to that expected for the oxidation and hydrolysis of Fe2+ displaced from clay interlayers, taking into account the buffering properties of the sediment matrix. This work shows that wetland acidification can occur in low sulfur-containing wetlands and is not exclusively a problem associated with sulfidic sediments.