Visualization of soil aggregates via X-ray fluorescence nanoscopy provides new insights into primary aggregation processes induced by litter inputs

One of the key parameters defining the healthy functioning of soil is its structure. The organization of mineral particles, organic matter (OM), water, and air into a complex matrix of soil aggregates plays a particularly important role in long-term carbon (C) storage, as C compounds can be ‘hidden’ within the aggregate structure, shielding them from decomposers. Soil aggregation is a dynamic process influenced by physical, chemical, and biological factors; however, the individual and combined effects of these factors on the formation and turnover of aggregates are not well understood.

The aim of this study was to examine the incorporation of fresh litter inputs with differing physicochemical properties, including their carbon-to-nitrogen (C/N) ratio—maize (C/N = 12) and straw (C/N = 103)—into aggregates formed de novo from mineral soil, with or without the presence of microbiota. Using rare-earth element oxides, we labeled structures formed during a four-week incubation with a single litter type and traced their incorporation into newly formed aggregates after mixing the soils and incubating them for a subsequent seven-week period.

We found that, regardless of quality, litter was the most important factor driving soil aggregation during the initial stages of the process. The presence of both litter types together further enhanced aggregate formation. Contrary to our hypothesis, and likely due to the short time frame of the experiment, neither microbial abundance nor community composition significantly affected overall aggregation. However, further visualization of the different litter-associated structures across the cross-sections of the aggregates from various size fractions using synchrotron radiation-based X-ray fluorescence nanospectroscopy (SR-nanoXRF) enabled us to estimate potential influence of the microbes via their preferred litter type. Specifically, contrary to our expectations the bulk analysis showed that bacteria-favoured low C/N ratio maize litter had a stronger effect on both overall aggregation and the formation of macroaggregates, which we initially hypothesized would be supported by the high C/N ratio straw litter preferred by fungi. However, further analysis of the XRF intensity maps confirmed an increasing incorporation of straw-associated soils into >250 μm structures, likely facilitated by fungal growth and hyphal enmeshing. Phospholipid fatty acid analysis further corroborated this, showing a relatively higher abundance of fungi in macroaggregates in straw-containing soil.

We also implemented semi-variogram analysis on the XRF maps, which allowed us to estimate the size and distribution of straw- and maize-associated structures within the aggregates. We found that while microaggregates were more commonly formed from individual litter-associated structures, larger aggregates (> 250 μm) were newly made from de-aggregated soil.

In conclusion, our study provides insights into the initial stages of aggregate formation following litter additions and the development of associated microbial communities. The spatial analysis enabled by SR-nanoXRF allowed us to visualize internal aggregate structures, shedding light on processes that cannot be fully understood through bulk analysis alone.