Submicron scale imaging of soil organic matter dynamics using NanoSIMS – From single particles to intact aggregates

Abstract

The specific features of the nano-scale secondary ion mass spectrometry (NanoSIMS) technology with the simultaneous analysis of up to seven ion species with high mass and lateral resolution enables us to perform multi-element and stable isotope measurements at the submicron scale. To elucidate the power of this technique, we performed an incubation experiment with soil particles of the fine silt and clay fractions (from an Albic Luvisol), with occluded particulate organic material and intact soil aggregates (from a Haplic Chernozem), using a ¹³C and ¹⁵N labelled amino acid mixture as tracer. Before and during 6-day incubation after the addition of the label, samples were consecutively prepared for NanoSIMS analysis. For this purpose, two different sample preparation techniques were developed: (i) wet deposition and (ii) the sectioning of epoxy resin embedded samples. Single soil particles (fine silt/clay fraction) showed an enrichment of ¹³C and ¹⁵N after label addition that decreased over time. On aggregates of particulate organic matter, re-aggregated during the 6-day incubation experiment, we could show a spatially heterogeneous enrichment of ¹³C and ¹⁵N on the particle surface. The enrichment in ¹⁵N demonstrated the diffusion of dissolved organic matter into intact soil aggregate interiors. The prospects of NanoSIMS for three dimensional studies of stable C and N isotopes in organo-mineral associations is demonstrated by the recorded depth profiles of the organic matter distribution on mineral particles.

Introduction

The solid phase of soils consists of a complex mixture of inorganic and organic components which are intimately associated with each other. Major inorganic components are quartz, clay minerals, (hydr)oxides of Fe, Mn and Al and carbonates. The organic phase is comprised of partially recalcitrant substances composed of fresh and decomposed materials derived from plant litter and faunal and microbial biomass. During soil formation, these components interact in such a way that primary soil particles are glued together forming micro-aggregates which are then bound together to macro-aggregates (Tisdall and Oades, 1982, Oades, 1984). As such, soils are highly complex materials that are structurally heterogeneous across a wide range of spatial and temporal scales (Oades and Waters, 1991, Herrmann et al., 2007b, Totsche et al., 2010). These soil processes of stabilization of organic matter (i.e. the formation of organo-mineral associations and the concurrent occlusion of organic particles in aggregated soil structures (von Lützow et al., 2006) occur at the sub-micron scale. To unravel the occlusion of soil organic matter (SOM) in aggregates as well as the formation of organo-mineral associations, an in situ analysis of soil particles could gain more precise information than destructive physical or chemical fractionation techniques. To investigate the evolution and composition of submicron-sized organo-mineral associations and aggregate interiors, novel micro-analytical techniques are required which allow the simultaneous analysis of the spatial distribution of elements involved in stabilization processes (for example C, N, Si, Al and Fe). This will allow a major step forward in the understanding of soil formation with significant implications for our conceptual understanding of the soil C and N cycles, structural stability and sorptive properties (von Lützow et al., 2008, Totsche et al., 2010).

Secondary ion mass spectrometry (SIMS) and time of flight-SIMS (TOF-SIMS) were used previously to visualize microbial activity (Cliff et al., 2002, DeRito et al., 2005, Pumphrey et al., 2009) in artificial soil systems (Si wafer as mineral matter model) and ex situ soil suspensions. A first attempt to track the fate of freshly added organic matter (OM) as ¹³C labelled plant material in soil aggregates was recorded with SIMS (Blair et al., 2006), although the lateral resolution and the recovery of the labelled plant material was low.

The nano-scale secondary ion mass spectrometry (NanoSIMS) is the latest generation of SIMS instruments allowing the simultaneous analysis of up to seven ion species (Cameca, NanoSIMS 50 L) with high sensitivity and a lateral resolution of up to 50 nm (using Cs⁺ as primary ion species and on samples with very low topography). As demonstrated before, NanoSIMS is an unprecedented tool for the analysis of biogeochemical processes and properties of soils (Herrmann et al., 2007a, Herrmann et al., 2007b, Clode et al., 2009). A beam of primary ions (Cs⁺ or O⁻) bombards a sample surface resulting in the release of secondary ions, which are then filtered and collected by a magnetic mass analyser. With Cs⁺ primary ions, negatively charged secondary ions, for example ¹²C⁻, ¹³C⁻, ¹²C¹⁴N⁻, ¹²C¹⁵N⁻ and ²⁸Si⁻, may be collected with a lateral resolution of up to 50 nm; with O⁻ primary ions, positively charged secondary ions, for example ¹²C⁺, ²⁸Si⁺, ²³Na⁺, ³⁹K⁺ and ⁴⁰Ca⁺, are detected with a lateral resolution of up to 150 nm. Mass resolution is considerably improved in comparison with previous SIMS generations and differentiation between the mass of ¹³C¹⁴N⁻ (27.016 amu) and the mass of ¹²C¹⁵N⁻ (27.009 amu) is feasible (Lechene et al., 2006). Consequently, the NanoSIMS enables us to investigate the elemental and isotopic composition of soils at the submicron scale. Nevertheless, the high sensitivity and lateral resolution of the NanoSIMS instrument can only sufficiently be used with appropriate sample preparation. Topography, outgassing and charging effects are especially of major concern to achieve high precision (Winterholler et al., 2008).

Until today, the NanoSIMS technique has been mainly applied in the field of material science, cosmochemistry, geology, mineralogy and biology (Hoppe, 2006, Lozano-Perez et al., 2008, Orphan and House, 2009, Wagner, 2009). In the field of soil science, the possible applications of NanoSIMS for the study of biogeochemical processes was reviewed by Herrmann et al. (2007b). The authors indicated the great potential for submicron studies of soil samples with a special focus on soil fauna, but also mentioned the potential for the study of soil organic matter stabilization. Using NanoSIMS, Herrmann et al. (2007a) were able to identify bacteria in the soil matrix. For this application, the authors used Pseudomonas fluorescens enriched in ¹⁵N, which they added to a soil sample that was subsequently dried and resin-embedded for NanoSIMS analysis. Another NanoSIMS study on a soil matrix was done by Clode et al. (2009), studying the nutrient uptake of roots. The authors were able to record the ${}^{15}{\text{NH}}_4^{+}$ uptake in microorganisms and in the intracellular sphere of the root cells. In the present study, we demonstrate the applicability of the NanoSIMS technique for research questions related to soil biogeochemistry. The focus on the present study lays therefore on the analysis of soil particles rather than on the visualization of soil organisms than microbial activity. We aim to study main elemental composition (C, N, Si, O) and the fate of isotopically enriched OM on soil compartments at a submicron scale, reaching from primary mineral particles to intact soil aggregates.

We chose a fine silt/clay mixture (<6.3 μm) from an Albic Luvisol for the investigation of micro-aggregates and small occluded particulate organic matter (oPOM_small, <20 μm) from a Haplic Chernozem for studying POM in soils. The samples were chosen in such a way to represent two important SOM pools in relation to the long term stabilization of soil organic carbon (SOC). The mineral associated SOM represents the largest reservoir of soil organic carbon, stabilised by the interaction with clay minerals and pedogenic oxides on decadal to centennial timescales (Christensen, 2001, von Lützow et al., 2008) The highly aromatic oPOM_small (high black carbon content) represents a SOM pool that is stabilized by its chemical recalcitrance (high aromaticity) but also by spatial inaccessibility (low bioavailability for microbes and enzymes) due to its occlusion in soil aggregates (Wagai et al., 2009). At the same time, clay sized mineral and small black carbon particles represent very important material for the sorption of nutrients and also contaminants. The chosen samples can therefore act as a reference for further micro-scale studies dealing with the stabilization of SOM and the sorption and desorption of contaminants.

All samples were incubated for 6 days with a ¹³C and ¹⁵N labelled amino acid mixture, and samples were consecutively prepared for NanoSIMS analysis during this period. The intact soil aggregates were used to study the diffusion of freshly added dissolved organic matter into the intact aggregate interior. With this technique, we were able to follow the isotopic enrichment and distribution of ¹³C and ¹⁵N on particle surfaces and of ¹⁵N within an intact soil aggregate. The focus in these studies is on the imaging of the spatial heterogeneity of C and N and their isotopes at the submicron scale, indicating spatial differences in SOM stabilization and utilization at soil microsites. We finally present a depth profile of SOM (as ¹²C¹⁴N⁻ secondary ion) on primary particles, demonstrating the possibility to study SOM in three dimensions at a submicron scale.