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Environmental Processes
Soil organic carbon stabilization: Mapping carbon speciation from intact microaggregates Maria Hernandez-Soriano, Ram Dalal, Frederick J. Warren, Peng Wang, Kathryn Green, Mark James Tobin, Neal W Menzies, and Peter M Kopittke Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03095 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018
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Soil organic carbon stabilization: Mapping carbon speciation from intact
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microaggregates
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Maria C. Hernandez-Soriano1, Ram C. Dalal1, Frederick J. Warren2, Peng Wang1,3, Kathryn
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Green4, Mark J. Tobin5, Neal W. Menzies1, Peter M. Kopittke1,*
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1
8
Queensland, 4072, Australia
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2
The University of Queensland, School of Agriculture and Food Sciences, St. Lucia,
The University of Queensland, Centre for Nutrition and Food Sciences, Queensland Alliance
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for Agriculture and Food Innovation, St. Lucia, Queensland, 4072, Australia
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3
12
Nanjing, Jiangsu, 210009, China
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4
14
Queensland, 4072, Australia
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5
Nanjing Agricultural University, College of Resources and Environmental Sciences,
The University of Queensland, Centre for Microscopy and Microanalysis, St. Lucia,
Australian Synchrotron, Clayton, Victoria, 3168, Australia
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*Corresponding author: Peter M. Kopittke,
[email protected], +61 7 3346 9149
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Abstract
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The clearing of land for agricultural production depletes soil organic carbon (OC) reservoirs,
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yet despite their importance, the mechanisms by which C is stabilized in soils remain unclear.
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Using synchrotron-based infrared microspectroscopy, we have for the first time obtained in
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situ, laterally-resolved data regarding the speciation of C within sections taken from intact
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free microaggregates from two contrasting soils (Vertisol and Oxisol, 0-20 cm depth)
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impacted upon by long-term (up to 79 y) agricultural production. There was no apparent
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gradient in the C concentration from the aggregate surface to the interior for any of the three
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forms of C examined (aliphatic C, aromatic C, and polysaccharide C). Rather, organo-mineral
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interactions were of critical importance in influencing overall C stability, particularly for
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aliphatic C, supporting the hypothesis that microaggregates form through organo-mineral
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interactions. However, long-term cropping substantially decreased the magnitude of the
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organo-mineral interactions for all three forms of C. Thus, although organo-mineral
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interactions are important for OC stability, C forms associated with the mineral phases are
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not entirely resistant to degradation. These results provide important insights into the
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underlying mechanisms by which microaggregates form, and the factors influencing the
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persistence of OC in soils.
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TOC
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INTRODUCTION
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Carbon (C) reservoirs in soil (ca. 2,344 Gt in the surface 3 m) exceed those in both the
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atmosphere (850 Gt) and the biotic pool (560 Gt) combined.1 However, the conversion of
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land from native vegetation to long-term agricultural cropping often reduces soil organic C
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(SOC) stocks by 20-60%.2-4 This marked depletion of SOC stocks results in the release of
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greenhouse gases. Given that the soil represents a large C pool, only comparatively small
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changes in SOC stocks can have important impacts on greenhouse gas concentrations. Not
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only does the decrease in SOC in long-term agricultural systems increase greenhouse gas
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emissions, it also results in the loss of soil quality and fertility, with land use change
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influencing both the content and composition of soil organic matter (SOM) with concomitant
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changes in the chemical and biological processes that determine soil functioning.5
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Accordingly, to both improve soil fertility and decrease release of greenhouse gases, it is
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important to optimize the management of SOM and to develop strategies for stabilizing C in
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soils.1, 6, 7 This has recently been recognized in the ‘soil carbon 4 per mille’ initiative which
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has an aspiration to increase global soil OC stocks by 4 per 1000 (0.4 %) per year as a
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compensation for global emissions of greenhouse gases.8
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In order to effectively understand and predict the potential sequestration of C in soils, it is
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first necessary to understand the underlying mechanisms of C stabilization and processing.7, 9,
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10
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mechanisms by which organic C is stabilized in soils. It has traditionally been assumed that
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the stability of SOC is regulated by the (i) preservation of complex (recalcitrant) compounds
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resistant to microbial degradation6 (ii) formation of organo-mineral associations11 and (iii)
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physical (spatial) inaccessibility of occluded SOC to microbial communities and enzymes.12-
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14
Indeed, despite its importance, there remains considerable uncertainty regarding the
The overall relative importance of each of these three underlying mechanisms to the overall
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stabilization of OC across a broad range of soils remains somewhat uncertain, with
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investigation of these mechanisms being the focus of the present study (for example, see 15).
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It is important to note that the currently-available evidence generally does not appear to
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support the first of these three hypotheses [i.e. that recalcitrant compounds, “humic
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substances”, form during the degradation process],7, 14, 16, 17 and this hypothesis cannot
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account for the long-term stabilization of otherwise labile SOC in soils.17 Nevertheless, it is
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known that the degree of ‘recalcitrance’ is important in the early stages of decomposition,
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although ‘spatial inaccessibility’ and ‘organo‐mineral interactions’ are important during the
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later stages of decomposition.18
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Previously, progress in identifying and confirming these underlying processes that regulate
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the cycling of SOC has been hindered by a lack of suitable approaches, such as those that
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provide in situ, laterally-resolved analyses of SOC composition within intact soil
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microaggregates. Previously, scanning electron microscopy coupled with energy-dispersive
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X-ray spectroscopy (SEM-EDS) has been found to be useful in this regard.19 Similarly, other
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approaches such as nanoscale secondary ion mass spectrometry (NanoSIMS) and µ-
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tomography are being used increasingly to investigate the cycling of OC in soils.20, 21 It has
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been shown comparatively recently that synchrotron-based spectroscopy [such as scanning
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transmission X-ray microspectroscopy (STXM) and infrared (IR) microspectroscopy] can
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provide laterally-resolved information on the molecular organization of SOC,13, 22, 23 the
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physical protection in the soil particles, and the co-localization of C sources with microbial
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processes.12, 13, 24 However, we are unaware of any studies that have provided laterally-
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resolved analyses of SOC speciation from entire, intact sections of microaggregates from
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soils impacted upon by long-term agricultural production. Such information is important in
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understanding the factors influencing the stability and turnover of organic C in soil systems.
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In the present study, we have utilized two paired-sites (Oxisol and Vertisol) from subtropical
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Australia to compare undisturbed soils to adjacent soils that have been used for agricultural
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production for either 60 y (Oxisol) or 79 y (Vertisol). The SOC content of these soils has
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been reduced markedly by agricultural production, with the decrease in SOC being 85% for
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the Oxisol (with the corresponding reduction in C stock being from 26 to 7 Mg C/ha) and
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76% for the Vertisol (with the corresponding reduction in C stock being from 61 to 10 Mg
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C/ha) (0–20 cm depth). Comparable C losses (up to 75%) have been previously reported for
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long-term (> 50 y) cultivation in tropical and sub-tropical systems.25-27 For the two soils, we
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used in situ, laterally-resolved synchrotron-based analyses to examine changes in the
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speciation and distribution of SOC using thin-sections obtained from free intact
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microaggregates (250-53 µm). For the first time using in situ, laterally-resolved analyses, our
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results link long-term changes in organo-mineral associations of various forms of C with the
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decline of SOC stocks – this information being crucial in understanding the mechanisms
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which influence SOC stability and cycling. It is important to note that it was not the aim of
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our study to perform a statistically-based experiment on land use change effects based on
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replicated samples, but rather to highlight the great potential of synchrotron-based methods to
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elucidate land-use change effects on important soil properties at the aggregate and sub-
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aggregate scale. We hypothesized that the sections taken from intact microaggregates would
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show a distinct gradient from the interior to the exterior of the microaggregates resulting from
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the occlusion of organic C by the mineral phase during microaggregate formation.
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Furthermore, we hypothesized that there would be a strong correlation between the clay
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mineral phase and the various forms of SOC due to the importance of organo-mineral
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associations.
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MATERIALS AND METHODS
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Soil collection and general analyses
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We collected two soils (Oxisol and Vertisol) from land-use pairs at two locations in
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Queensland (Australia). The two soils were selected as they differ markedly in their
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properties, including mineralogy (see later). For the Oxisol (26° 42’ S, 151° 48’ E), the native
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vegetation was ‘vine scrub’, while for the Vertisol (26° 48’ S, 150° 54’ E), the dominant
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native vegetation was brigalow (Acacia harpophylla). The Oxisol has an average annual
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rainfall of 770 mm, with a yearly average maximum temperature of 24.7 °C and average
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minimum of 11.4 °C. The Vertisol has an average annual rainfall of 610 mm, with a yearly
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average maximum temperature of 26.9 °C and average minimum of 11.9 °C. For both sites,
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the rainfall is summer-dominant and the climate classified as sub-tropical. The paired sites for
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both the Oxisol and the Vertisol encompass two land-use types < 100 m apart, being
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undisturbed soil (native vegetation) and cropped soil. The conversion of native vegetation to
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agricultural cropping occurred 60 y ago for the Oxisol and 79 y ago for the Vertisol. The
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Oxisol has mainly been used for cropping of peanut (Arachis hypogaea) and maize (Zea
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mays), while the Vertisol has mainly been used for winter cropping of wheat (Triticum
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aestivum). Both soils are low input systems. For the Oxisol, the soils received no fertilizer for
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the first 55 y of cropping, with fertilizer for the last 5 y applied as 50 kg/ha/y as diammonium
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phosphate. For the Vertisol, soils received only 30 kg N/ha/y for 51 y, with 100 kg N/ha/y
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and 10 kg P/ha/y since this time for 28 y. No organic amendments (such as manures) have
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been applied at either site. For the cropping soils, cultivation consisted of two or three
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operations per year with a chisel plough (approximately 20 cm depth) to control weeds during
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fallow.
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Soil samples were collected from both land-uses (undisturbed and cropping) at the two sites
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from a depth of 0-20 cm. The samples were collected in the October-November period (mid-
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spring), with those from the cropping soils collected either at post-harvest (Vertisol) or at
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maturity (Oxisol). Three individual replicate samples were collected, sealed in plastic
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containers, and stored at 4° C until further analysis. For background analyses, soils were first
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sieved to 2 mm and air-dried. Soil pH was measured using 1:5 soil:water suspensions with
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air-dried soil. For organic C, we measured concentrations in the bulk soil plus four different
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size fractions that were obtained by wet sieving,28 being > 2000 (large macroaggregates),
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2000-250 (small macroaggregates), 250-53 (free microaggregates), and < 53 µm (silt+clay).
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The free water-stable microaggregates (250-53 µm) accounted for 5.3-9.6 % of the total
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aggregates in the undisturbed soils and increased to 8.9-13 % in the cropped soils, although
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occluded microaggregates are also present within macroaggregates. Organic C concentrations
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were measured by dry combustion using a CN analyzer (VarioMax) after oven-drying (60
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°C). Particle size analysis was performed using the pipet method.29 Mineralogy was
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determined for the undisturbed soils using X-ray diffraction.
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Infrared microspectroscopy
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For infrared microspectroscopy, subsamples of the three air-dried individual replicates were
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combined to form a composite sample. A composite sample was used as only limited samples
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could be examined using synchrotron-based infrared microspectroscopy. Soils were sieved (2
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mm), and intact microaggregates particles (53–250 µm in diameter) were isolated by wet
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sieving28 from both land uses of the two soil types. The use of this approach resulted in the
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collection of free water-stable microaggregates rather than microaggregates occluded within
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macroaggregates.28 The free water-stable microaggregates (53–250 µm in diameter) were
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then air-dried until needed. Approximately 20-30 typical example free microaggregates (53–
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250 µm in diameter) were selected, placed on filter paper, moistened gently through
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humidification.22 Once moistened, the free microaggregates were frozen at -20 °C and semi-
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thin sections of microaggregates were cut using a diamond knife without embedding media
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using a cryo-ultramicrotome. The sections were cut with the ultramicrotome set to a thickness
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of 200 nm, although the actual thickness of each section was not measured. Sections were
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then transferred to Cu grids (carbon free, 200 mesh, silicon monoxide coating) in order to
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allow the sections to dry and be transferred to the Australian Synchrotron (below).
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The sections were removed from the Cu grids and analyzed at the IR Microspectroscopy
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beamline at the Australian Synchrotron (Melbourne, Australia), using a Bruker Hyperion
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2000 infrared microscope and V80v Fourier transform infrared (FT-IR) spectrometer. Due to
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time constraints, only a single replicate section from each of the four treatments could be
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examined. The microscope was equipped with 36× (0.5 numerical aperture) condenser and
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objective optics, a narrowband mercury cadmium telluride detector, and a detection aperture
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was selected to sample an area of 5 µm × 5 µm. The maps (5 µm step size over ca. 150 µm ×
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150 µm) were obtained from 64 co-added scans (4 cm-1 resolution) and the spectra were
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acquired in transmission mode. A single map (150 µm × 150 µm) was obtained for each of
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the four sections collected from the intact microaggregates.
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Spectral maps were processed using the software OPUS 7.2 (Bruker Optik GmbH, Germany).
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Map profiles were created for absorbance at 3630 cm-1 (O–H groups of clays), 2920 cm-1
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(aliphatic-C), 1600 cm-1 (aromatic-C), and 1035 cm-1 (polysaccharides-C).22, 30 The peak at
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3630 cm-1 corresponds to the stretching vibrations of O-H groups of clay minerals, the peak
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at 2920 cm-1 corresponds to the C-H stretching vibrations of aliphatic biopolymers, the peak
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at 1600 cm-1 corresponds to C=C stretching of aromatic C or N-H deformations, and the peak
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at 1035 cm-1 corresponds to C-O stretching vibrations of polysaccharide C.30 In each case the
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integrated area under the absorbance peak was applied to the map, selecting appropriate
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baseline points either side of the absorbance peak [3550-3740 cm-1 (O–H groups of clays),
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2800-3000 cm-1 (aliphatic C), 1500-1750 cm-1 (aromatic C), and 950-1170 cm-1
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(polysaccharide C)].
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The spectra collected for each map were examined and a linear regression (Genstat v16) used
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to examine the correlation between the amount of clay (absorbance at 3630 cm-1) and either
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polysaccharides C (absorbance at 1035 cm-1), aromatic C (absorbance at 1600 cm-1) or
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aliphatic C (absorbance at 2920 cm-1).22, 30 The R2 coefficients and regression slopes were
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used as indices for associations, the former indicating the residual variability around the
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association while the latter the relative strength of association. Based upon the signal to noise
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ratio, it was estimated that these synchrotron-based analyses were ca. 100-fold more sensitive
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than conventional (globar source) IR microspectroscopy (also see Miller and Dumas 31 for a
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comparison of a synchrotron source and a globar source).
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Bulk infrared spectroscopy
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Soil samples were sieved (2 mm) and finely ground using a mortar and pestle. FTIR-ATR
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spectra were collected for all soil samples using a PerkinElmer Spectrum 100 FTIR
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spectrometer equipped with a deuterated triglycine sulfate (DTGS) detector. The ATR
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accessory (PerkinElmer UATR) was equipped with a diamond crystal. Background spectra
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were collected every five samples and subtracted from each recorded spectrum. For each
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spectrum, 16 scans were co-added at a resolution of 4 cm-1 from 4000 to 650 cm-1. The
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spectra were vector-normalized in order to minimize intensity variations between the spectral
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data set.
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RESULTS
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Undisturbed soils
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Using synchrotron-based IR microspectroscopy, we obtained laterally-resolved information
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on C speciation [polysaccharide C (1035 cm-1), aromatic C (1600 cm-1), aliphatic C (2920
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cm-1), and clay minerals (3650 cm-1)]22, 30 and distribution in thin sections taken from entire,
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intact microaggregates of the Oxisol and Vertisol (Table 1 and Figure 1). Giving
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consideration firstly to the undisturbed soils, it was observed that the distribution of the
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various C forms was not homogenous, but rather, they were distributed as discrete areas of C
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accumulation in the microaggregate section both in the interior and the edges of the
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microaggregate sections (see top panels of Figure 2 and Figure 3). For these undisturbed
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soils, it was apparent that there were relationships between the distribution of clay minerals
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and the various forms of C. Indeed, correlation analysis (which indicates co-localization)
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identified that aliphatic-C was closely associated with clay minerals in both the undisturbed
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Oxisol (R2 = 0.97) and Vertisol (R2 = 0.72) (Figure 2 and Figure 3). For the two other forms
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of C (polysaccharide C and aromatic C), whilst relationships with clay minerals were
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generally strong in the Oxisol (R2 values 0.83 and 0.77), relationships with clay minerals in
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the Vertisol were poor (R2 values 0.29 and 0.22) (Figure 2 and Figure 3). Thus, whilst some
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of the polysaccharide C and aromatic C was associated with clay minerals, a higher
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proportion of this C was associated with areas spatially unrelated to clay minerals. It is
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possible that this poorer relationship for the Vertisol was due to its lower C concentration
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(Table 1), with not all reactive sites being occupied.
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It was also noted that there was no apparent gradient in accumulation of the various C forms
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from the interior to the exterior of the microaggregate, as might occur if organic C was
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surrounded (occluded) in a central core by clay minerals, for example. To examine this more
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closely, changes in the various forms of organic C and clay minerals were examined across a
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transect through the middle of the microaggregate section. For both the Oxisol (Figure 4) and
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the Vertisol (Figure 5), there was no clear evidence of a distinct trend in organic C, nor was
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there a clear core of organic C surrounded by clay minerals.
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Vertisol
Oxisol 800
Undisturbed Cropped
600
-1
Aggregates (g kg soil)
Undisturbed Cropped
400
200
0 ates ates ilt+clay ates ates ilt+clay ates ates S S greg aggreg aggreg greg aggreg aggreg g g a a icro icro acro l macro acro l macro M M m m e e al al Sm Sm Larg Larg
245 246
Figure 1. Distribution of aggregate size fractions in undisturbed and cropped soils, from an
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Oxisol (left) and Vertisol (right).
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Table 1. Soil pH, clay content, concentration of organic carbon (g OC kg soil -1) in bulk soil and soil size fractions, being > 2000, 2000-250,
250
250-53, and < 53 µm. pH (1:5
Sand
Silt
Clay
water)
(%)
(%)
(%)
Mineralogy*
C/N
OC (g kg
ratio
soil-1) Bulk soil
Oxisol
Vertisol
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Undisturbed
5.2
55
8
36
Cropped
5.7
62
8
30
Undisturbed
6.9
31
18
51
Cropped
7.9
34
16
50
Q, K, H
Q, S, K
OC (g kg aggregate-1)
> 2000
2000-250
250-53
µm
µm
µm
< 53 µm
13.8
52
53
47
56
46
11.5
7.9
7.3
6.7
8.1
20
11.4
22
21
21
16
19
12.2
5.3
5.7
5.0
3.4
7.0
* Listed in decreasing order. Abbreviations: Q, quartz; H, hematite; K, kaolinite; S, smectite.
252
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Figure 2. Semi-thin (200 nm) sections of microaggregates (