Investigating the role of stoichiometry as an influence on soil phosphorus content and forms

Abstract

The elemental composition (stoichiometry) of soil organic matter (SOM) plays an important role in the dynamics of nutrient transformations in terrestrial ecosystems. Many previous studies have reported several factors affect the stoichiometry of SOM (e.g. climate, land use, soil properties) and confirmed a relatively constrained stoichiometry of carbon (C) and nitrogen (N). On the other hand, correlations of C and N to phosphorus (P) in SOM are usually much weaker. This can partly be attributed to the fact that soils contain substantial quantities of inorganic as well as organic P. Some studies have reported stronger correlations of organic P than total P to the key elements of SOM (i.e. C and N), though are still not as strong as between C and N (mostly present as organic N). There are multiple possible reasons for this, including limitations in the way organic P is measured and the diversity of organic P forms that may be present in soils. Recent advances in solution ³¹P NMR (nuclear magnetic resonance) analysis provide an opportunity to reassess the stoichiometry of P in SOM, as NMR provides a detailed and quantitative assessment of the various classes of organic P present in soils. Thus the implementation of solution ³¹P NMR analysis facilitates investigation of whether the weak overall stoichiometric relationships of P in SOM may be masking stronger stoichiometric control of particular SOM components or chemical forms. These issues are agronomic and environmental importance because organic phosphorus represents a substantial pool of P in soils. Although organic P is not directly plant available, it can become available to plants through microbial mineralisation. Understanding the processes involved would be of potential benefit to agricultural producers in assessing P fertility of soils and better estimating P fertiliser requirements and also to environmental managers in assessing the risk of P transfer from soils to water ways where excess P is a major cause of eutrophication. This thesis describes a range of activities aimed at improving understanding the role of stoichiometry of P in SOM. The main limitation of solution ³¹P NMR spectroscopy as a method for analysis of soil P is the inherently low sensitivity of NMR. This results in long acquisition times (typically one day per sample) and hence low sample throughput. Furthermore, low sensitivity limits detection and quantification of species present in low concentrations. The first part of this thesis reports on efforts to improve sensitivity by tightening the ratio of soil to solution in the extraction step preceding NMR analysis. The most commonly used procedure involves extraction with a mixture of NaOH and EDTA at a ratio of 1:20. A range of tighter extraction ratios down to 1:4 were tested for a set of four Red Chromosol topsoils with low P contents, and it was shown that at lower extraction ratios the signal to noise ratio of spectra was improved with little or no loss in extraction efficiency, there was no loss in signal resolution and no difference in the distribution of P species detected. Thus it can be confidently claimed that, at least for soils similar to those tested, employing an extraction ratio of 1:4 provides a sensitivity benefit with no detrimental effects on resolution or quantitation. Using the improved NMR methodology, a wider set of twenty Red Chromosol soils were analysed and a range of P forms quantified in the extracts. A novel P pool structure was proposed based on strong correlations among sets of P species present. A set of four pools was proposed: (i) orthophosphate, (ii) humic P, (iii) cellular organic P (the sum of lipid P, RNA P, diester P and pyrophosphate) and (iv) inositol phosphate P (the sum of myo- and scyllo-inositol hexakisphosphate). In terms of stoichiometry, it was shown that the cellular organic P pool had closer relationships than other P pools with C and N. These findings support the overall hypothesis that some SOM pools would exhibit stronger P stoichiometry than other pools. These findings also raise the question of whether the cellular P pool is closely related to the microbial biomass P pool or a plant residue P pool. In part, to address the possible correspondence of the cellular P pool with plant residues, physical fractionation (fine fraction < 50 μm and coarse fraction > 50 μm) of the twenty Red Chromosol soils was carried out and the elemental composition (C, N, P) of the fractions determined. Carbon concentrations were found to correlate strongly with N in both size fractions. In contrast, C concentrations correlated moderately (r²=0.37, p <0.01) with P in the coarse but not in the fine fractions. Again, these results support the overall hypothesis that some SOM pools would exhibit stronger P stoichiometry than other pools. In particular, since the organic matter in the coarse fraction isolated in this manner, which is often referred to as particulate organic carbon (POC), is usually presumed to be dominated by plant residues, these results suggest stronger stoichiometric constraint of the plant-dominated fraction of soil organic matter. Solution ³¹P NMR analysis was carried out on a small selection of fractionated soils and this indicated consistent differences in organic P composition between the fractions, with the fine fractions containing a larger proportion of humic P and the coarse fraction containing relatively more cellular P. Unfortunately, practical constraints relating to the amount of soil available and the low sensitivity of NMR analysis restricted the number of soil fractions that could be analysed in this way. The final set of experiments described in this thesis addressed the potential connection between the cellular organic P pool and the microbial biomass P (MBP) pool. These experiments involved incubation of a pasture soil rich in organic matter with an easily assimilated source of carbon (glucose). Glucose addition resulted in the expected increase in soil respiration, but the effects on measured soil P pools were unexpected. The traditional measure of the MBP pool – the difference in resin P between fumigated and unfumigated soils was only slightly higher for the glucose amended soil, although closer inspection of the components of this measurement indicated a complex response in which resin P was strongly depleted on glucose amendment but fumigation failed to release all of the extra P that had been taken up. Solution ³¹P NMR analysis clearly showed that glucose addition did not significantly increase the concentration of cellular organic P. The implication is that the cellular P pool detected by NMR is not closely related to established methods of determining MBP. The results presented in this thesis provide insight into the role of stoichiometry as a control on the P content of SOM. Overall, the hypothesis that weak overall stoichiometric control of P in SOM masks stronger stoichiometric control of particular components or chemical forms was sustained. In particular, amongst chemical pools, P stoichiometry was strongest for a pool identified as cellular organic P and amongst physical pools, P stoichiometry was stronger for the coarse fraction than the fine fraction. The use of combinations of techniques (e.g. NMR and size separation) and comparison of techniques (e.g. NMR and traditional measures of MBP) was important in providing this new insight and this general approach should be pursued further to better understand the complex transformations that control the fate of P in soils.